Common variants of Drosophila melanogaster Cyp6d2 cause camptothecin sensitivity and synergize with loss of Brca2

Abstract

Many chemotherapeutic agents selectively target rapidly dividing cells, including cancer cells, by causing DNA damage that leads to genome instability and cell death. We used Drosophila melanogaster to study how mutations in key DNA repair genes affect an organism's response to chemotherapeutic drugs. In this study, we focused on camptothecin and its derivatives, topotecan and irinotecan, which are type I topoisomerase inhibitors that create DNA double-strand breaks in rapidly dividing cells. Here, we describe two polymorphisms in Drosophila Cyp6d2 that result in extreme sensitivity to camptothecin but not topotecan or irinotecan. We confirmed that the sensitivity was due to mutations in Cyp6d2 by rescuing the defect with a wild-type copy of Cyp6d2. In addition, we showed that combining a cyp6d2 mutation with mutations in Drosophila brca2 results in extreme sensitivity to camptothecin. Given the frequency of the Cyp6d2 polymorphisms in publcly available Drosophila stocks, our study demonstrates the need for caution when interpreting results from drug sensitivity screens in Drosophila and other model organisms. Furthermore, our findings illustrate how genetic background effects can be important when determining the efficacy of chemotherapeutic agents in various DNA repair mutants.

Keywords: chemotherapeutics; cytochrome p450; double-strand breaks; homologous recombination.

Figure 1

The scpt mutant is sensitive to camptothecin but not to topotecan or ionizing radiation. (A) Camptothecin sensitivity of the scpt mutant as well as two null alleles of brca2. All data points represent three independent trials of five to six vials each. (B) Topotecan sensitivity of the scpt mutant and brca2^KO mutant. All data points represent three independent trials of five to six vials each. (C) Ionizing radiation sensitivity of the scpt mutant as well as two brca2 mutant alleles. All data points represent two to four independent trials of one bottle each. SDs are shown as error bars.

Figure 2

Meiotic mapping of the scpt mutation. (A) Cross scheme for meiotic mapping. Flies that inherit the white⁺ marker have red eyes. Flies that inherit two mutant copies of the scpt mutation do not survive treatment with 25 µM camptothecin. The percentage of surviving flies with red eyes should be equal to the recombination distance between the white⁺ marker and scpt. (B) Multiple meiotic mapping crosses identified a region containing the scpt mutation. Both DrosDel deficiencies (ED) and P{SUPor-P} elements (KG) were used. Distances on the x-axis are approximations of the physical distance between these markers. The region shown spans from cytological band 55C to 60F (the terminal quarter of chromosome 2R, or about 7 megabases).

Figure 3

Mutations in Cyp6d2 affect transcription and RNA processing. (A) Diagram of the Cyp6d2 gene structure. Protein coding regions are represented by black boxes, whereas gray bars represent introns and UTRs. Dotted line indicates the region shown in B. Primers shown were used in RT-PCRs in C−E. (B) Nucleotide and amino acid alignment of wild-type (WT) and scpt mutant Cyp6d2. scpt SNPs in this region are underlined. The heme-binding cysteine is boxed in the WT protein. Failure to splice intron three (sequence in lower-case letters) results in a frameshift mutation and a premature stop codon (*). Total wild-type protein length is 512 amino acids. (C−E) Semiquantitative RT-PCR of wild-type and mutant larvae. Primers indicated below gels refer to those shown in (A). RT-PCR with (+RT) and without (−RT) reverse transcriptase was performed. Genomic (Gen) DNA template is also shown for comparison. Sizes of molecular weight markers are indicated. (C) The XInt3/F2 primer pair yields a PCR product of 709 bp if splicing of intron 3 is correct. The rp49 primer pair (control reaction) yields PCR products of 398 bp and a genomic product of 460 bp. (D) The XInt2/R1 primer pair yields a PCR product of 375 bp if introns 2 and 3 are properly spliced. Intron 3 length is 70 bp. (E) XInt1/F1 yields a PCR product of 440 bp if intron 1 is correctly spliced.

Figure 4

Rescue of camptothecin sensitivity by a single wild-type Cyp6d2 transgene. All data points represent three to four independent trials of five to six vials each. Error bars represent SDs. (A−B) Rescue of both cyp6d2 mutants. A precise excision of P{GT1}CG42565 was used as the cyp6d2^NT mutant in (B). (C) Partial rescue of the brca2⁴⁷ allele.