Chromosome segregation and organization are targets of 5′-Fluorouracil in eukaryotic cells

Abstract

The antimetabolite 5 0 -Fluorouracil (5FU) is an analog of uracil commonly employed as a chemotherapeutic agent in the treatment of a range of cancers including colorectal tumors. To assess the cellular effects of 5FU, we performed a genome-wide screening of the haploid deletion library of the eukaryotic model Schizosaccharomyces pombe. Our analysis validated previously characterized drug targets including RNA metabolism, but it also revealed unexpected mechanisms of action associated with chromosome segregation and organization (post-translational histone modification, histone exchange, heterochromatin). Further analysis showed that 5FU affects the heterochromatin structure (decreased levels of histone H3 lysine 9 methylation) and silencing (down-regulation of heterochromatic dg/ dh transcripts). To our knowledge, this is the first time that defects in heterochromatin have been correlated with increased cytotoxicity to an anticancer drug. Moreover, the segregation of chromosomes, a process that requires an intact heterochromatin at centromeres, was impaired after drug exposure. These defects could be related to the induction of genes involved in chromatid cohesion and kinetochore assembly. Interestingly, we also observed that thiabendazole, a microtubule-destabilizing agent, synergistically enhanced the cytotoxic effects of 5FU. These findings point to new targets and drug combinations that could potentiate the effectiveness of 5FU-based treatments.

Hypomodified tRNA in evolutionarily distant yeasts can trigger rapid tRNA decay to activate the general amino acid control response, but with different consequences

“…pombe trm8Δ mutants in YES and EMMC media at 38°C and 39°C, expression of both tY(GUA) and tP(AGG) was required to completely suppress the growth defects in YES + glycerol media ( S7 Fig ). By contrast, overexpression of tY(GUA) and tP(AGG) had no effect on the known temperature sensitivity of trm8Δ mutants in YES media containing 5-FU [ 61 , 62 ] ( S7 Fig ), perhaps due to reduced levels of Ψ and 5-methyluridine modifications, which could trigger decay of other hypomodified tRNA species in trm8Δ mutants.…”

“…Although tY(GUA) overexpression almost completely restored growth of S. pombe trm8Δ mutants in YES and EMMC media at 38˚C and 39˚C, expression of both tY(GUA) and tP (AGG) was required to completely suppress the growth defects in YES + glycerol media (S7 Fig). By contrast, overexpression of tY(GUA) and tP(AGG) had no effect on the known temperature sensitivity of trm8Δ mutants in YES media containing 5-FU [61,62]…”

Predicting chemotherapeutic drug combinations through gene network profiling

“…We recently performed a screen for genes that confer doxorubicin resistance (DXR) using fission yeast ( Schizosaccharomyces pombe ) as a model, and found that a network of genes protected cells against drug cytotoxicity 24 25 . We noted that many cells with mutations in these DXR genes exhibited hypersensitivity to a range of drugs, not specifically doxorubicin 23 26 27 , and this pointed to a potential overlap in drug resistance mechanisms and/or the existence of a genetic network counteracting multiple cytotoxic agents in fission yeast.…”

“…In this work, we employed a serial dilution spotting assay, a commonly used method, which can be varied by streaking log-phase cells or by measuring changes in cell density 23 30 56 57 58 . Unlike previous studies, we have uniquely incorporated a computation of dose response, which, in our view, increases the accuracy of the assay.…”

“…To provide evidence for the utility of such a work flow, we interrogated our previously identified doxorubicin resistance (DXR) genetic network and the intersection of doxorubicin with six other drugs: hydroxyurea (HU), an inhibitor of the deoxyribonucleotide biosynthesis enzyme ribonucleotide reductase (RNR) 15 ; methyl methanesulfonate (MMS), an alkylating agent that causes nucleotide base damage and impedes the progress of the DNA replication fork 16 ; camptothecin (CPT), a topoisomerase I inhibitor 17 ; thiabendazole (TBZ), a microtubule destabilizing agent 18 ; cisplatin, an intra-DNA strand cross-linker and blocker of transcription 19 20 and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor 21 . With the exception of TBZ, which targets mitotic spindle microtubules, the remaining drugs are reported to induce DNA aberrations that cause DNA damage 15 16 17 18 19 20 21 22 23 .…”