Genomic screening in vivo reveals the role played by vacuolar H+ ATPase and cytosolic acidification in sensitivity to DNA-damaging agents such as cisplatin

Mol Pharmacol. 2007 Feb;71(2):416-25. doi: 10.1124/mol.106.030494. Epub 2006 Nov 8.

Abstract

Screening the Saccharomyces cerevisiae homozygous diploid deletion library against a sublethal concentration of cisplatin revealed 76 strains sensitive to the drug. As expected, the largest category of deletions, representing 40% of the sensitive strains, was composed of strains lacking genes involved in DNA replication and damage repair. Deletions lacking function of the highly conserved vacuolar H+ translocating ATPase (V-ATPase) composed the category representing the second largest number of sensitive strains. The effect on cell death exhibited by V-ATPase mutants was found to be a general response to various DNA damaging agents as opposed to being specific to cisplatin, as evidenced by sensitivity of the mutants to hydroxyurea (a DNA-alkylating agent) and UV irradiation. Loss of V-ATPase does not affect DNA repair, because double mutants defective for V-ATPase function and DNA repair pathways were more sensitive to cisplatin than the single mutants. V-ATPase mutants are more prone to DNA damage than wild-type cells, indicated by enhanced activation of the DNA damage checkpoint. Vacuole function per se is not cisplatin-sensitive, because vacuolar morphology and vacuolar acidification were unaffected by cisplatin in wild-type cells. V-ATPase also controls cytoplasmic pH, so the enhanced sensitivity to DNA damage may be associated with the drop in pHi associated with V-ATPase mutants. The increased loss in cell viability induced by cisplatin at lower pH in V-ATPase mutants supports this hypothesis. The loss in viability seen in wild-type cells under the same conditions was far less dramatic.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antineoplastic Agents, Alkylating / pharmacology
  • Cisplatin / pharmacology*
  • Cytosol / chemistry*
  • DNA Damage / drug effects
  • DNA Repair / genetics
  • DNA Replication / genetics
  • Drug Resistance
  • Gene Library
  • Genome, Fungal*
  • Hydrogen-Ion Concentration
  • Saccharomyces cerevisiae / genetics
  • Sequence Deletion
  • Vacuolar Proton-Translocating ATPases / physiology*

Substances

  • Antineoplastic Agents, Alkylating
  • Vacuolar Proton-Translocating ATPases
  • Cisplatin