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Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase

View Article: PubMed Central - PubMed

ABSTRACT

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotidereductase, leading to lowered cellular pools of deoxyribonucleosidetriphosphates. The reduced levels for DNA precursors is believed to causereplication fork stalling. Upon treatment of the hyperthermophilic archaeonSulfolobus solfataricus with HU, we observe dose-dependentcell cycle arrest, accumulation of DNA double-strand breaks, stalled replicationforks, and elevated levels of recombination structures. However,Sulfolobus has a HU-insensitive class II ribonucleotidereductase, and we reveal that HU treatment does not significantly impactcellular DNA precursor pools. Profiling of protein and transcript levels revealsmodulation of a specific subset of replication initiation and cell divisiongenes. Notably, the selective loss of the regulatory subunit of the primasecorrelates with cessation of replication initiation and stalling of replicationforks. Furthermore, we find evidence for a detoxification response induced by HUtreatment.

No MeSH data available.


Related in: MedlinePlus

Effects of Chronic and Acute Treatment of Sulfolobussolfataricus with Hydroxyurea(A) Serial dilutions of S. solfataricus cells were plated onmedia containing the indicated concentrations of HU.(B) Viable cell counts measured by plating efficiency following the indicateddoses of HU; assays were performed in triplicates and the bars indicate themean; error bars are SDs. Results are expressed relative to untreated cells (setat 100%).(C) Flow cytometry profiles of cells following the indicated treatment andrecovery times.(D) Agarose gel analysis of the integrity of genomic DNA isolated from cellsfollowing exposure to 5 or 10 mM HU for 4 or 7 hr.(E) Representative micrograph showing phase contrast and fluorescence imaging oftreated or untreated cells. DNA was stained with DAPI and membranes withFM-464.
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Figure 1: Effects of Chronic and Acute Treatment of Sulfolobussolfataricus with Hydroxyurea(A) Serial dilutions of S. solfataricus cells were plated onmedia containing the indicated concentrations of HU.(B) Viable cell counts measured by plating efficiency following the indicateddoses of HU; assays were performed in triplicates and the bars indicate themean; error bars are SDs. Results are expressed relative to untreated cells (setat 100%).(C) Flow cytometry profiles of cells following the indicated treatment andrecovery times.(D) Agarose gel analysis of the integrity of genomic DNA isolated from cellsfollowing exposure to 5 or 10 mM HU for 4 or 7 hr.(E) Representative micrograph showing phase contrast and fluorescence imaging oftreated or untreated cells. DNA was stained with DAPI and membranes withFM-464.

Mentions: First, we plated serial dilutions of Sulfolobussolfataricus P2 on gelrite plates containing increasingconcentrations of HU. As seen in Figure 1A,chronic exposure to 5 mM HU completely inhibited growth. Next, we sought todetermine the effect of acute exposure to HU on cell survival. We incubatedexponentially growing S. solfataricus cells with 5 or 10 mM HUfor 4 or 7 hr (Figure 1B). HU treatment wasclearly toxic to cells with 66% cells remaining viable after 4 hr treatment with5 mM HU, dropping to 24% after 7 hr exposure. 10 mM HU showed a stronger effectwith 32% and 17% cells viable after 4 and 7 hr, respectively.


Hydroxyurea-Mediated Cytotoxicity Without Inhibition ofRibonucleotide Reductase
Effects of Chronic and Acute Treatment of Sulfolobussolfataricus with Hydroxyurea(A) Serial dilutions of S. solfataricus cells were plated onmedia containing the indicated concentrations of HU.(B) Viable cell counts measured by plating efficiency following the indicateddoses of HU; assays were performed in triplicates and the bars indicate themean; error bars are SDs. Results are expressed relative to untreated cells (setat 100%).(C) Flow cytometry profiles of cells following the indicated treatment andrecovery times.(D) Agarose gel analysis of the integrity of genomic DNA isolated from cellsfollowing exposure to 5 or 10 mM HU for 4 or 7 hr.(E) Representative micrograph showing phase contrast and fluorescence imaging oftreated or untreated cells. DNA was stained with DAPI and membranes withFM-464.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5134839&req=5

Figure 1: Effects of Chronic and Acute Treatment of Sulfolobussolfataricus with Hydroxyurea(A) Serial dilutions of S. solfataricus cells were plated onmedia containing the indicated concentrations of HU.(B) Viable cell counts measured by plating efficiency following the indicateddoses of HU; assays were performed in triplicates and the bars indicate themean; error bars are SDs. Results are expressed relative to untreated cells (setat 100%).(C) Flow cytometry profiles of cells following the indicated treatment andrecovery times.(D) Agarose gel analysis of the integrity of genomic DNA isolated from cellsfollowing exposure to 5 or 10 mM HU for 4 or 7 hr.(E) Representative micrograph showing phase contrast and fluorescence imaging oftreated or untreated cells. DNA was stained with DAPI and membranes withFM-464.
Mentions: First, we plated serial dilutions of Sulfolobussolfataricus P2 on gelrite plates containing increasingconcentrations of HU. As seen in Figure 1A,chronic exposure to 5 mM HU completely inhibited growth. Next, we sought todetermine the effect of acute exposure to HU on cell survival. We incubatedexponentially growing S. solfataricus cells with 5 or 10 mM HUfor 4 or 7 hr (Figure 1B). HU treatment wasclearly toxic to cells with 66% cells remaining viable after 4 hr treatment with5 mM HU, dropping to 24% after 7 hr exposure. 10 mM HU showed a stronger effectwith 32% and 17% cells viable after 4 and 7 hr, respectively.

View Article: PubMed Central - PubMed

ABSTRACT

In many organisms, hydroxyurea (HU) inhibits class I ribonucleotidereductase, leading to lowered cellular pools of deoxyribonucleosidetriphosphates. The reduced levels for DNA precursors is believed to causereplication fork stalling. Upon treatment of the hyperthermophilic archaeonSulfolobus solfataricus with HU, we observe dose-dependentcell cycle arrest, accumulation of DNA double-strand breaks, stalled replicationforks, and elevated levels of recombination structures. However,Sulfolobus has a HU-insensitive class II ribonucleotidereductase, and we reveal that HU treatment does not significantly impactcellular DNA precursor pools. Profiling of protein and transcript levels revealsmodulation of a specific subset of replication initiation and cell divisiongenes. Notably, the selective loss of the regulatory subunit of the primasecorrelates with cessation of replication initiation and stalling of replicationforks. Furthermore, we find evidence for a detoxification response induced by HUtreatment.

No MeSH data available.


Related in: MedlinePlus