Hydroxyurea Induces Cytokinesis Arrest in Cells Expressing a Mutated Sterol-14α-Demethylase in the Ergosterol Biosynthesis Pathway

Abstract

Hydroxyurea (HU) has been used for the treatment of multiple diseases, such as cancer. The therapeutic effect is generally believed to be due to the suppression of ribonucleotide reductase (RNR), which slows DNA polymerase movement at replication forks and induces an S phase cell cycle arrest in proliferating cells. Although aberrant mitosis and DNA damage generated at collapsed forks are the likely causes of cell death in the mutants with defects in replication stress response, the mechanism underlying the cytotoxicity of HU in wild-type cells remains poorly understood. While screening for new fission yeast mutants that are sensitive to replication stress, we identified a novel mutation in the erg11 gene encoding the enzyme sterol-14a-demethylase in the ergosterol biosynthesis pathway that dramatically sensitizes the cells to chronic HU treatment. Surprisingly, HU mainly arrests the erg11 mutant cells in cytokinesis, not in S phase. Unlike the reversible S phase arrest in wild-type cells, the cytokinesis arrest induced by HU is relatively stable and occurs at low doses of the drug, which likely explains the remarkable sensitivity of the mutant to HU. We also show that the mutation causes sterol deficiency, which may predispose the cells to the cytokinesis arrest and lead to cell death. We hypothesize that in addition to the RNR, HU may have a secondary unknown target(s) inside cells. Identification of such a target(s) may greatly improve the chemotherapies that employ HU or help to expand the clinical usage of this drug for additional pathological conditions.

Heme deficiency sensitizes yeast cells to oxidative stress induced by hydroxyurea

“…Understanding the new cellkilling mechanisms of a well established small molecule drug with multiple clinical implications is of great importance for the development of novel therapeutics. We have recently shown that in the presence of sterol deficiency, HU causes cell lethality mainly by stably arresting S. pombe cells in cytokinesis (15). As we show in our next paper, 3 when applied with the inhibitors of heme or sterol biosynthesis, the cell-killing effect of HU can be significantly enhanced in S. pombe and S. cerevisiae, as well as in the opportunistic fungal pathogen Candida albicans.…”

“…Interestingly, these mutants are not killed by aberrant mitosis or DNA damage that are commonly observed in DRC mutants but by previously unknown mechanisms. In a previous report, we showed that HU induces cytokinesis arrest in cells carrying a hypomorphic mutation in the erg11 gene, which encodes the enzyme sterol-14␣-demethylase required for sterol biosynthesis (15). In this study, we report the identification of a novel hem13-1 mutant in the heme biosynthesis pathway.…”

“…We have previously reported our characterization of one of the newly screened hus mutant erg11-1 (15). Here, we report our results with the second screened mutant that carries a novel mutation in hem13 whose gene product is the predicted enzyme coproporphyrinogen III oxidase required for the biosynthesis of heme (17) (Fig.…”

“…In contrast, HU and SMP had only a minimal effect on heme levels in the hem13-1 cells, which is probably because heme was already at a basal level required for cell growth. Heme is known to be required for ergosterol biosynthesis, and abundant ergosterol is crucial for HU resistance in S. pombe (15,46). However, the exact mechanism by which HU decreases the heme level inside wild-type cells remains unknown.…”

“…The specificities of the antibodies were verified as described in previous studies (15,23,59). The Rad3-dependent phosphorylation of Chk1 was assessed using the mobility-shift method developed by the Russell lab using the mouse monoclonal anti-HA 12CA5 antibody (Sigma) (60).…”