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Studies of Intracellular Transport and Anticancer Drug Action by Functional Genomics in Yeast
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes.

A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway.

The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells.

In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2008. , 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 402
Keyword [en]
5-fluorouracil, tRNA modifications, Saccharomyces cerevisiae, carbamoyl phosphate synthase, V-ATPase
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-9408ISBN: 978-91-554-7360-0OAI: oai:DiVA.org:uu-9408DiVA: diva2:172959
Public defence
C10:301, BMC, Husargatan 3, Uppsala
Opponent
Supervisors
Available from: 2011-03-04 Created: 2008-11-25 Last updated: 2011-10-28Bibliographically approved
List of papers
1. Functional genomics of monensin sensitivity in yeast: Implications for post-Golgi traffic and vacuolar H+-ATPase function
Open this publication in new window or tab >>Functional genomics of monensin sensitivity in yeast: Implications for post-Golgi traffic and vacuolar H+-ATPase function
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2008 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 280, no 3, 233-248 p.Article in journal (Refereed) Published
Abstract [en]

We have screened a complete collection of yeast knockout mutants for sensitivity to monensin, an ionophore that interferes with intracellular transport. A total of 63 sensitive strains were found. Most of the strains were deleted for genes involved in post-Golgi traffic, with an emphasis on vacuolar biogenesis. A high correlation was thus seen with VPS and VAM genes, but there were also significant differences between the three sets of genes. A weaker correlation was seen with sensitivity to NaCl, in particular rate of growth effects. Interestingly, all 14 genes encoding subunits of the vacuolar H(+)-ATPase (V-ATPase) were absent in our screen, even though they appeared in the VPS or VAM screens. All monensin-sensitive mutants that could be tested interact synthetically with a deletion of the A subunit of the V-ATPase, Vma1. Synthetic lethality was limited to mutations affecting endocytosis or retrograde transport to Golgi. In addition, vma1 was epistatic over the monensin sensitivity of vacuolar transport mutants, but not endocytosis mutants. Deletions of the two isoforms of the V-ATPase a subunit, Vph1 and Stv1 had opposite effects on the monensin sensitivity of a ypt7 mutant. These findings are consistent with a model where monensin inhibits growth by interfering with the maintenance of an acidic pH in the late secretory pathway. The synthetic lethality of vma1 with mutations affecting retrograde transport to the Golgi further suggests that it is in the late Golgi that a low pH must be maintained.

Keyword
endosome, monensin, V-ATPase, vacuole, vesicular transport
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-97866 (URN)10.1007/s00438-008-0359-9 (DOI)000258540900005 ()18612650 (PubMedID)
Available from: 2008-11-25 Created: 2008-11-25 Last updated: 2011-03-17Bibliographically approved
2. Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast
Open this publication in new window or tab >>Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast
2008 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, Vol. 14, no 4, 666-674 p.Article in journal (Refereed) Published
Abstract [en]

We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38 degrees C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.

Keyword
5-fluorouracil, pseudouridine synthase, tRNA methylase, tRNA modifications, yeast
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-97867 (URN)10.1261/rna.966208 (DOI)000254256800007 ()18314501 (PubMedID)
Available from: 2008-11-25 Created: 2008-11-25 Last updated: 2009-11-12Bibliographically approved
3. Identification of genes whose overexpression confer resistance to the anticancer drug 5-fluorouracil in yeast
Open this publication in new window or tab >>Identification of genes whose overexpression confer resistance to the anticancer drug 5-fluorouracil in yeast
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:uu:diva-97868 (URN)
Available from: 2008-11-25 Created: 2008-11-25 Last updated: 2011-01-12Bibliographically approved

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