Genomic screening in vivo reveals the role played by vacuolar H+ ATPase and cytosolic acidification in sensitivity to DNA-damaging agents such as cisplatin.
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.
Liao
Chunyan
C
Pharmaceutical Science Research Division, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK.
Hu
Bin
B
Arno
Matthew J
MJ
Panaretou
Barry
B
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology*; D003600:Cytosol / Q000737:chemistry*; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004351:Drug Resistance; D015723:Gene Library; D016681:Genome, Fungal; D006863:Hydrogen-Ion Concentration; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017384:Sequence Deletion; D025262:Vacuolar Proton-Translocating ATPases / Q000502:physiology*
D018906:Antineoplastic Agents, Alkylating; D025262:Vacuolar Proton-Translocating ATPases; D002945:Cisplatin