ToxSci Advance Access originally published online on May 22, 2007
Toxicological Sciences 2007 99(2):522-531; doi:10.1093/toxsci/kfm123
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Transgenic Rescue of Methotrexate-Induced Teratogenicity in Drosophila melanogaster
Department of Biology, Biosciences, Queen's University, Kingston, Ontario K7L 3N6, Canada
1 To whom correspondence should be addressed at Department of Biology, Bioscience Complex, Room 2522, Queen's University, Kingston, Ontario K7L 3N6, Canada. Fax: (613) 533-6617. E-mail: walkervk{at}biology.queensu.ca.
Received April 6, 2007; accepted May 7, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The folic acid analog methotrexate (MTX), a competitive inhibitor of dihydrofolate reductase (DHFR), is used to treat a variety of cancers and autoimmune disorders. However, MTX also causes a wide range of toxic effects in healthy cells and is an established teratogen. Efforts to "rescue" the defects caused by MTX by administering a folate analog or by transgenic expression of a DHFR with an altered affinity for MTX have been attempted in a variety of mammals but limited protection was conferred. As a result, our understanding of the effect of MTX at the molecular genetic level remains incomplete and, in addition, continued mammalian sacrifice is not ideal. Due to the similarity of teratogenic effects produced by MTX in Drosophila melanogaster these insects were transformed with DHFR alleles to determine if rescue could be achieved. The resulting "MTX-resistant" flies were subsequently used to investigate changes in gene expression in response to MTX using semiquantitative reverse transcription PCR. The majority (12/14) of key transcripts that were affected in MTX-exposed females including transcripts involved in cell cycle, defense response, and transport were "rescued" in the "MTX-resistant" transgenic flies. These studies illustrate the utility of this invertebrate model for the investigation of molecular effects of MTX-induced teratogenicity, MTX-resistant DHFRs for gene therapy techniques, and teratogenic protection.
Key Words: methotrexate; dihydrofolate reductase; Drosophila melanogaster; teratogenesis; rescue; gene expression.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Dihydrofolate reductase (EC 1.5.1.3, DHFR) converts 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), which in turn, acts as a cofactor in one-carbon transfer reactions and is essential for the biosynthesis of purines, thymidylate, and several amino acids (Hooijberg et al., 2003
). Thus, inhibition of DHFR can prevent cell proliferation and eventually lead to cell death. Therapies using antifolates, such as the mammalian antifolate methotrexate (MTX), exploit this characteristic and are used to treat leukemia, osteosarcoma, breast, head, neck, and bladder cancers (McGuire, 2003) as well as other diseases such as ectopic pregnancy (Lozeau and Potter, 2005), rheumatoid arthritis, psoriasis, systemic lupus (Zimecki and Artym, 2004), Crohn's disease (Sun and Das, 2005), asthma (Corrigan et al., 2005), and in combination with misoprostol is used to induce abortion (Creinin et al., 2003).
Although MTX is the most commonly used chemotherapeutic agent for the aforementioned cancers and its use for treatment of other diseases is steadily increasing, MTX is toxic and can produce embryonic lethality and other severe teratogenic effects (Lloyd et al., 1999; Ostensen, 2004). In humans, the teratogenic phenotype can include craniofacial and skeletal abnormalities, as well as pulmonary, cardiac, and gastrointestinal organ malformations (Aviles et al., 1991; Bawle et al., 1998). Similar MTX-induced teratogenesis has been observed in rats (Vinson and Hales, 2002), mice (Darab et al., 1987), rabbits (DeSesso and Goeringer, 1991), and cats (Khera, 1976).
There are devastating effects of MTX exposure during development but its obvious value as a therapeutic agent has promoted attempts to "rescue" fetuses from antifolate induced teratogenicity. Leucovorin, a structural analog of THF, has been shown to alleviate some of the morphological defects in rabbits (DeSesso and Goeringer, 1991). When "drug-resistant" DHFRs were used for transplant (May et al., 1995) or constitutively expressed in transgenic embryos or placental tissues of mice (Sutton et al., 1998) limited protection against MTX exposure was conferred. However, our understanding of the effect of MTX at the molecular genetic level remains incomplete. In addition, there have been no studies that examine prolonged exposure to MTX or if the effects can be ameliorated at high drug doses.
Many pathways and enzymes are conserved between mammals and the fruit fly Drosophila melanogaster (Reiter and Bier, 2002), and as a result this model invertebrate has been used to study a variety of "human diseases" including cancer (Potter et al., 2000), cardiac disease (Bier and Bodmer, 2004), aging (Grotewiel et al., 2005; Saitoe et al., 2005), and neurodegeneration (Bonini and Fortini, 2003). Drosophila also shows MTX toxicity (Affleck et al., 2006b) and has potential as a model for MTX "rescue therapies." Mutations in Drosophila DHFR, isolated by cell line selection (L30Q) or by molecular modeling (L22R), produce a MTX-resistant phenotype when expressed in Chinese hamster ovary cells (Affleck et al., 2006a; Neumann et al., 2003). Here we test the hypothesis that maternal expression of these mutant DHFR alleles will prevent MTX-mediated teratogenesis in transgenic Drosophila. Insight into these toxicity rescue experiments was also furthered by the examination of transcript levels or key, sentinel genes.
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METHODS AND MATERIALS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Plasmid construction.
Wild-type (GenBank #U06861) Drosophila Dhfr cDNA in pcDNA3.0 (Neumann et al., 2003
) was digested with BglII and SacII restriction endonucleases (Fermentas, Burlington, ON) and the Dhfr ORF subsequently subcloned into pINDY5 designated pINDY5Dhfr. Primer sequences L22R (5'-GGC ATC AGA GGC GAT CGA CCA TGG CGC ATT AAA TC-3') and L30Q (5'-CGCAATAAATCTGAGCAGAAGTACTTCAGCCGCACC-3'), with underlined nucleotides designed for the appropriate amino acid substitutions, were used with the pINDY5Dhfr template to create L22R and L30Q Drosophila Dhfr mutations. A QuickChange Site-directed Mutagenesis kit (Stratagene, La Lolla, CA) was used according to the manufacturer's instructions and the resulting constructs were designated pINDY5L22R and pINDY5L30Q, respectively, after confirmation by sequencing.
P-element mediated transformation.
Female Drosophila w1118 (white-eyed) were placed in cups with egg collecting media (2% agar, 5% sucrose, and 
0.01% neutral red) and a small amount of live brewer's yeast. After overnight acclimation, the cups were changed every 30 min and after three or more cup exchanges, eggs were collected from non yeasted cups and aligned on microscope slides. Embryos were injected at the posterior end with an Eppendorf Femtojet microinjector (Eppendorf, Westbury, NY) with pINDY5Dhfr or pINDY5L30Q at five times the concentration of p
2,3 helper DNA for a total DNA concentration of 0.4 µg/µl. pINDY5L22R was injected by Genetic Services, Inc. (Sudbury, MA) according to their protocols.
Surviving G0 adults were collected after eclosion and crossed with w1118 flies. Transgenic lines were subsequently crossed to balancer-bearing w1118 flies (T(2:3) SSaD/CyO, In(3LR)TM3, Sb) and the chromosome that carried the insert was identified. If possible, homozygous lines were established (Socolich, 2003). Genomic DNA was isolated from homozygous transgenic lines and the complete insertion of the transgene was confirmed by sequencing with flanking pINDY5 primers. Two independent transgenic lines for each construct were retained for further analysis.
Dhfr transcript abundance.
Offspring of control wild-type Canton S (CS; wt), w1118, and transgenic pINDY5Dhfr, pINDY5L22R, and pINDY5L30Q flies were crossed to a line of flies bearing an ubiquitous GAL4 transcriptional activator, daughterless-Gal4 (daGal4; Wodarz et al., 1995). Approximately, 30 female F1 progeny were collected and RNA was isolated using Trizol RNA isolation (Bogart and Andrews, 2005). Total RNA (10 ng) was used for each semiquantitative reverse transcription PCR (qRT-PCR) reaction using QuantiTect SYBR Green RT-PCR kit (Qiagen, Mississauga, ON), using primers and methods as previously described (Affleck et al., 2006b). Primers (LP 5'-AGCGCGGTTACTCTTTCACCAC-3' and RP 5'-GTGGCCATCTCCTGCTCAAAGT-3') specific to endogenous D. melanogaster actin 5C (DmAct5C; K00667) were used to determine the levels of D. melanogaster control RNA. All qRT-PCR reactions were done in triplicate and the ratio of control (DmAct5C) to Dhfr messenger RNAs (mRNAs) was determined.
Western blotting.
Female progeny from crosses of CS, w1118, pINDY5Dhfr, pINDY5L22R, and pINDY5L30Q fly lines with daGal4 flies were collected. For 30 adults, 500 µl of CellLyticTM-M (Sigma-Aldrich, St Louis, MO) with 1x ethylenediaminetetraacetic acid-free protease inhibitor (Roche Diagnostics, Laval, QC) was used according to the CelLytic-M protein extraction protocol, with the addition of homogenization for 15 s using a Rotor Stator homogenizer (KikaWerk, Germany) prior to incubation with lysis buffer. Total protein concentration was determined by bicinchoninic assay protein assay (Pierce, Rockford, IL). Proteins (10 µg) from each sample were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) and blocked with 3% powdered milk in Tris-Buffered Saline Tween-20 (TBS-T). The membrane was incubated with rabbit anti-actin (1:2000; Sigma-Aldrich) and rabbit anti-DHFR (1:5000; Hao et al., 1994), in 1.5% powdered milk in TBS-T for 1 h. The membrane was then washed with TBS-T (3 x 30 s then 3 x 15 min), incubated with goat anti-rabbit IgG–horse radish peroxidase (HRP) (1:10,000; Life Technologies, Burlington, ON) for 1 h, and washed again in TBS-T. Goat anti-rabbit IgG-HRP was detected on Biomax MS film (Eastman Kodak Company, Rochester, NY) using the BM chemiluminescence kit (Boehringer Mannheim, Laval, QC) according to the manufacturers instructions for 10–30 s.
Fecundity and teratogenicity assays.
After crossing each of two lines of flies bearing each of the different genotypes with daGal4 driver flies (as described above for western analysis) female progeny were collected within 6 h of eclosion. These females were crossed with 6- to 12-h posteclosion CS males (three females; two males) and placed on medium (2.2% yeast, 5% molasses, 5.3% cornmeal, 2.2% agar, 0.1% methyl-p-hydroxybenzoic acid, 0.4% propionic acid) containing 0, 0.01, 0.1, 1, or 10µM MTX (triplicate assays for all concentrations and genotypes). Fresh food was provided every 12 h and the number of eggs laid/female/day at each MTX concentration was determined over 21 days. The morphology of oviposited eggs was examined on day 3 or 4 using a Zeiss Axioplan 2 microscope (Zeiss, Germany) at 10x optical zoom and images were captured using Axiovision 4.5 software (Zeiss).
Females derived from crosses of the different lines with the daGal4 driver lines that had been exposed to MTX-containing medium for 13 days were transferred to regular (no MTX) medium at the end of day 13 and fecundity determined as described above for days 14–21.
Additional crosses were undertaken as described above for each of the transgenic lines, and female progeny from these crosses were crossed with CS males and dissected at days 2, 3, 4, 5. The number of stage 9–14 (s9–14; large yolky) follicles/ovary was counted. Follicles were staged according to King (1970). A minimum of 20 ovaries per line were examined. The average number of s9–14 follicles per ovary (and standard deviations) was calculated for each fly line on each day.
As well as the daGal4 driver line, females were also collected from crosses of the different lines to two other driver lines (Manseau et al., 1997), CG3743 c306 (drives transgene expression in stalk and border cells) and CG3750 c535 (drives transgene expression in posterior pole s8–9 cells, central nervous system and later throughout the oocyte). These females were also placed on medium containing 0 and 1µM MTX and fecundity determined for days 1–6.
ANOVA statistical analysis using JMP statistical software (SAS Institute Inc., Cary, NC) was used to determine significant differences in oviposition and follicle numbers.
Ovarian morphology and transcript levels.
Transgenic lines were crossed with the daGal4 driver line as described and virgin female progeny were crossed with CS males (three females; two males). After exposure to medium supplemented with MTX (1µM) or control medium, females were dissected in 0.1M NaCl and their ovaries removed. Ovaries were also obtained on day 21 from similarly treated females that had been exposed to either 1µM MTX or 10µM MTX for 13 days, and then transferred to regular food from day 14–21. Ovaries were fixed in 25% glutaraldehyde, 0.1M sodium cacodylate, pH 7.4 for 8 h, washed in 0.1M sodium cacodylate overnight, and rinsed in fresh solution the following morning. Specimens were postfixed with 1% osmium tetroxide in 0.1M sodium cacodylate for 2 h, rinsed in distilled water and dehydrated with increasing concentrations of ethyl alcohol at 10 min intervals. They were then chemically dried using increasing concentrations of hexamethlydisilazane at 15-min intervals and subsequently air dried overnight. Once dried, ovaries were mounted on stubs and coated with a thin layer of gold palladium using a Hummer VI-A sputtering system (Anatech Ltd, Alexandria, VA) and scanning electron micrograph (SEM) images captured with a JEOL/JSM-840 (JEOL Ltd., Peabody, MA) SEM operated at 10 kV.
RNA was isolated from ovaries of control (untreated) and MTX-treated (1µM) female progeny derived from crosses of w1118 and pINDY5L30Q fly lines with daGal4 flies. Total RNA (50 ng) was used for each qRT-PCR reaction as previously described. Primers specific to sequences of previously identified transcripts that increased or decreased in abundance after exposure to MTX in S3 cells and/or in CS ovaries were used (Affleck et al., 2006b). All qRT-PCR reactions were done in triplicate and means and standard deviation determined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Transgene Expression
After verifying the presence of an intact transgene in each of the lines (not shown), Dhfr expression was assayed by examining the ratio of actin transcripts to Dhfr mRNAs by qRT-PCR. There was a similar modest, but significant increase in Dhfr mRNA levels in all transgenic lines, consistent with the additional Dhfr transgene (mean for all transgenic lines 1.5 ± 0.2 vs. 1 ± 0.0 for w1118; p < 0.05). Similarly, when protein extracts from the transgenic lines were assayed by western blots using anti-actin (a control for equal protein loading) and anti-DHFR antibodies, there was little difference in DHFR abundance in any of the transgenic lines (Fig. 1; data not shown).
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-actin and DHFR in representative fly lines. Extracts from w1118, pINDY5Dhfra, pINDY5L22Ra, and pINDY5L30Qa females were separated by gel electrophoresis (10 µg of total protein), transferred to a PVDF membrane and incubated with anti-actin (control for equal loading) and anti-DHFR. One representative gel of a total of eight gels is shown.Teratogenicity Assays
To determine if there was a differential susceptibility to MTX toxicity amongst the various tested genotypes, teratogenicity assays were performed for each transgene using two independently derived lines with insertions on different chromosomes (Table 1; results not shown). Since each pair of transgenic lines tested had the same response to MTX, independent of insert location, results from a single representative line are shown in the figures and used for statistical analysis. CS and w1118 control lines were used as controls. When exposed to a low dose of MTX (0.01 and 0.1µM), females from all lines oviposited a similar number of eggs each day over a 21-day period (data not shown). The number of eggs laid at low concentrations was not significantly different from the oviposition rate on medium containing no MTX (all ps > 0.05). However, when females were placed on 1µM MTX only those fly lines with the pINDY5L30Q mutant transgenes continued to oviposit over the 21 day period (Fig. 2). Egg numbers from pINDY5L30Q transgenic flies on 1µM MTX were not significantly different from sister transgenic flies placed on regular food without MTX (p > 0.05). Oviposition was completely arrested in the two controls and in all lines transformed with six different transgenes after day 5 when placed on medium containing 10µM MTX (data not shown).
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When the transgenic lines were crossed to the alternative driver lines (CG3743 c306 and CG3750 c535), similar to results observed when the daGal4 driver line was used as parents; only those fly lines with the pINDY5L30Q mutant transgene continued to oviposit after 5 days on 1µM MTX. For example, oviposition averaged five eggs per day and eight eggs per day on day 5 when transgene expression was regulated by CG3743 c306 and CG3750 c535, respectively (results not shown).
After 13 days, fly lines that were infertile after exposure to 1µM MTX were transferred to regular medium. All fly lines resumed oviposition; however, there was a lag in the egg laying rate, similar to the lag seen on days 1 and 2 with immature flies (Fig. 3). On day 16 and day 17 there was no difference in oviposition in w1118, pINDY5Dhfr, or pINDY5L22R when compared to their untreated sisters (all ps > 0.05). Likewise, there was no significant difference in egg numbers from CS or pINDY5L22R females on day 18 (p > 0.05). By day 19 there was no significant difference in the number of eggs laid by any of the CS, w1118, pINDY5Dhfr, and pINDY5L22R lines when compared to their sisters, which had not been exposed to MTX (all ps > 0.05). Significantly, control and transgenic females that were transiently exposed to very high concentrations (10µM) of MTX remained infertile, except for the pINDY5L30Q females. These flies showed some recovery (e.g., 1.5 eggs/day/female on days 18–21).
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When females generated from the same crosses and mated with CS males were dissected at days 2, 3, 4, and 5, the number of s9–14 follicles/ovary mirrored the egg counts. There was a significantly higher number of yolky follicles per ovary for pINDY5L30Q females on 1µM MTX compared to all other lines (Fig. 4; all ps < 0.05). Strikingly, by day 5, pINDY5L30Q females were the only females with yolky follicles (Fig. 4). There was no difference in any of the lines when unexposed to MTX (results not shown).
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Ovaries dissected on day 5 were visualized using SEM in order to examine ovary morphology in more detail. Ovaries from females on regular media appeared normal with follicles at all stages of development (Fig. 5a). Ovaries from all CS, w1118, pINDY5Dhfr, and pINDY5L22R females examined (
9 females/fly line) showed only small (< s9) follicles after 5 days of 1µM MTX exposure (Figs. 5b–e). In contrast, ovaries from pINDY5L30Q females (n = 9) on 1µM MTX appeared similar to ovaries (with s1–14 follicles) from their sisters on regular medium (Fig. 5f). Light microscope examination of eggs oviposited on day 3 or 4 by females on regular media were of normal appearance with large chorionic appendages (Fig. 6). Eggs oviposited by pINDY5L30Q females treated with 1µM MTX also appeared normal. However, a majority of eggs (10 eggs/fly line examined) oviposited by CS, w1118, pINDY5Dhfr, and pINDY5L22R females after 3 days, or all eggs after 4 days of 1µM MTX exposure, had semi-transparent chorions and small chorionic appendages (Fig. 6; data not shown).
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Eggs and ovaries from females that had been previously treated with 1µM MTX for 13 days and subsequently allowed to recover on regular medium were also examined. In all fly lines, 2 days after transfer to regular medium, a few eggs, all of which were small, semitransparent and with no or severely diminished chorionic appendages (data not shown) were observed. Subsequently, after 6–8 days all the lines showed full fecundity (Fig. 3) with eggs of normal size (
500 µm) and fully developed chorionic appendages (data not shown). Dissected ovaries on day 21 from nine females of each CS, w1118, pINDY5Dhfr, and pINDY5L22R (Fig. 7c) all had normal appearance with numerous follicles (s1–14), including large yolky follicles. Ovaries from nine pINDY5L30Q females that had been exposed to 10µM MTX for 13 days also showed some recovery (s1–14) and some yolky follicles (Fig. 7d). Ovaries dissected from sisters that had not been exposed to MTX (day 21) and those placed on medium containing 1µM MTX (day 5) were used for comparison (Figs. 7a and 7b).
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The Effect of MTX on Transcript Abundance
To determine if the morphological changes seen in 1µM MTX-treated ovaries reflected changes in transcript abundance, mRNAs from ovaries of w1118 and females expressing L30Q DHFR (pINDY5L30Qa) were examined using qRT-PCR. Most (five) of the transcripts previously shown to increase in abundance in response to MTX also increased after exposure to 1µM MTX in w1118 ovaries with levels of two transcripts (Fst and GstE9) not substantially increased (Table 2). However, there was little change in abundance for all seven mRNAs in pINDY5L30Q ovaries (Table 2). Similarly, transcript levels previously shown to decrease in abundance after MTX treatment were also shown to decline after exposure to 1µM MTX in w1118 ovaries (Table 3). Again, most (5/7) of these transcripts showed little change in abundance in the pINDY5L30Q ovaries (Table 3). Two transcripts (loki and Mcm6) were less abundant in ovaries from the MTX-treated pINDY5L30Q females compared to untreated controls, but the levels were not as depressed as those seen in w1118 ovaries.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONFUNDINGREFERENCES |
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MTX is a potent antifolate and thus it is perhaps not surprising that it is toxic and teratogenic (McGuire, 2003
). Attempts to "rescue" the disturbing side effects of antifolate therapy by expression of altered DHFRs with a reduced affinity for MTX have been undertaken in mice (May et al., 1995; Sutton et al., 1998; Zhao et al., 1997), but complete amelioration of the morphological abnormalities was not observed in all embryos (Sutton et al., 1998). Due to a growing concern over the use of mammalian sacrifice for toxicity and teratogenic testing, we propose that "rescue" treatment should be examined by gene expression in a nonmammalian system. Since altered Drosophila DHFRs have been shown to confer resistance in a mammalian cell line (Affleck et al., 2006a; Neumann et al., 2003) and maternal effects of MTX treatment have been shown in D. melanogaster (Affleck et al., 2006b), MTX-resistant fly lines could be a valuable model that could also serve to monitor accompanying changes of mRNA levels of key, MTX-indicator genes.
Teratogenic and Toxicity Rescue
Multiple lines of transgenic flies bearing altered Drosophila Dhfr cDNAs that confer high levels of MTX resistance to a mammalian cell line were generated. These included a DHFRL30Q mutation, derived from MTX-resistant Drosophila cells, which generated transgenic CHO cells that were able to proliferate at 50% of control growth in 1µM MTX (IC50 = 0.84µM), and a DHFRL22R mutation designed in silico that produced transgenic CHO cells that were 200-fold more resistant (IC50 > 100µM) than CHO cells expressing wild-type Drosophila DHFR (Affleck et al., 2006a). To ensure that any observed MTX resistance was not due to position effect, two lines, each with insertions on different chromosomes were analyzed for Dhfr transcript abundance and DHFR expression (Table 1). As anticipated, due to the presence of the endogenous Dhfr there was a slight increase in the overall Dhfr transcript abundance in all the transgenic lines. In no case did lines with an altered DHFR have higher than expected levels of Dhfr mRNA or DHFR than control transgenic flies with wild-type Drosophila DHFR (Table 1; Fig. 1; data not shown). Thus, any resistance observed in the transgenic fly lines should be due to the decreased affinity for MTX mediated by the transgene.
At low drug concentrations (0.01 and 0.1µM MTX) it is unlikely that folate pools are sufficiently reduced to affect oviposition and likely resulted in the observation that there were no differences in fecundity for either the control (CS or w1118) or transgenic (pINDY5Dhfr, pINDY5L22R, and pINDY5L30Q) lines. At higher concentrations (1µM MTX), however, the fecundity was significantly decreased in CS, w1118, pINDY5Dhfr, and pINDY5L22R females. pINDY5L30Q females, however, continued to oviposit for 21 days and CS, w1118, pINDY5Dhfr, and pINDY5L22R females allowed to recover from MTX treatment were able to lay eggs with a healthy phenotype after 6–8 days (Fig. 3; data not shown). At a very high dose (10µM MTX) all females were sterile after day 5 (data not shown) with only pINDY5L30Q showing recovery when the drug was removed (Fig. 7). Thus, the MTX dose-dependent response observed in murine embryos (Sutton et al., 1998) and in humans (Moisa et al., 2006) is also shown in our Drosophila model. In addition, our model shows that teratogenic risk is reduced after removal of MTX, similar to the reduced risk observed in human mothers four months after treatment with antifolate (Lloyd et al., 1999).
Transgenic females expressing the DHFRL22R mutant were as susceptible to MTX treatment as nontransgenic females. Murine L22R DHFR expression in transgenic mouse embryos provided some protection against MTX but the decrease in catalytic ability seen in this mutant DHFR was cited as the reason that MTX-induced teratogenicity was not completely ameliorated (Sutton et al., 1998). Recombinant Drosophila DHFRL22R shows such low activity that the Km could not be determined (Affleck et al., 2006a) suggesting that the enzyme binds neither DHF nor MTX at physiological concentrations. The fact that CHO cells showed a 200-fold increase in MTX resistance when transfected with this mutant gene emphasizes the importance of testing for MTX resistance in a whole organism where there is no option of slowing down cell proliferation until sufficient THF becomes available. Thus, the presence of this mutant allele was ineffective in rescuing MTX-induced abnormalities.
Of all the lines, only the L30Q mutant DHFR offered protection from MTX treatment, and this was reflected not only in egg counts and follicle counts but also in SEM observations of their ovaries (Fig. 5) that were morphologically similar irrespective of MTX exposure (0 or 1µM). Similarly, there was no evidence of teratogenesis in progeny eggs from pINDY5L30Q crosses, compared to the abnormal eggs observed prior to the cessation of oviposition from all the other lines (Fig. 6). The absence of teratogenesis suggests that the resistance allele must be expressed in ovarian tissues to effectively "rescue" developing oocytes. This is the case in lines crossed to the ubiquitous daGal4 driver, and this was confirmed using the CG3743 c306 and CG3750 c535 drivers that specifically direct expression of the transgene in ovaries (stalk and border cells or in pole cells, and follicle cells, respectively). Since pINDY5L30Q transgenic females with ovarian-specific drivers were unaffected by MTX treatment (1µM) and showed no evidence of teratogenesis, this rules out the possibility that MTX resistance might be mediated elsewhere, such as the digestive or circulatory system. Furthermore, these experiments indicate that L30Q DHFR was able reduce folate and as a result, DNA replication is likely uninhibited in these ovaries allowing the rapid increase in oocyte volume and protein production through to s14 of oogenesis.
"Rescue" of Transcripts for Other Loci
As well as being an effective inhibitor of DHFR, MTX perturbs levels of transcripts involved in defense, cell cycle, transport, signaling, and transcription (Affleck et al., 2006b). It is not known if these effects are a consequence of the decrease in THF or if MTX has additional targets. The availability of transgenic pINDY5L30Q females, which are "rescued" from the teratogenic effects of MTX allowed this question to be explored. Most of the 14 target transcripts that had previously been identified in Drosophila cell lines or ovaries (exposed to 0.05 and 10µM MTX, respectively) were also affected in 1µM MTX-treated w1118 ovaries. Only two stress response transcripts (GstE9 and Fst) were at levels similar to untreated controls, possibly due to the lower dose of MTX used in the present study. In contrast, however, when levels of the 14 mRNAs were quantified from pINDY5L30Q ovaries, they showed near concordance with control, untreated ovaries; 12/14 transcripts showed less than a 1.3-fold change (log2 ratio 
0.42), and only two transcripts, loki and Mcm6, had modestly lower transcript levels. Both of the genes corresponding to these transcripts are important for DNA fidelity; loki is likely a DNA damage checkpoint protein (Xu et al., 2001) and Mcm6 is associated with DNA replication initiation and chorion gene amplification (Claycomb et al., 2004). Both the chorion and chorionic appendages appeared normal in pINDY5L30Q embryos that had been maternally exposed to 1µM MTX (Fig. 6). Thus the sevenfold change (log2 ratio of – 2.8 ± 0.4) in the level of Mcm6 transcripts seen in females with the L30Q DHFR does not appear to effect morphology. However, the 446-fold change (log2 ratio of – 8.8 ± 0.5) in transcript levels in w1118 females, may contribute profoundly, giving rise to abnormal embryos and eventually complete sterility. The small, semi-transparent eggs with diminished chorionic appendages, which were oviposited by drug-treated w1118 females is suggestive of disruption of transcript titers in follicle cells, the layer of cells that encircle the developing oocyte. We speculate that the majority of transcripts showing near normal abundance in pINDY5L30Q ovaries such as the cell cycle, signaling, and transport mRNAs (Table 3) represent loci whose expression is "downstream" of Dhfr and that normal levels of these transcripts depend on adequate titers of THF.
As noted, there have been previous attempts to "rescue" mammals from the teratogenic effects of MTX using DHFRs with altered MTX affinities (May et al., 1995; Sutton et al., 1998); however, this is the first time "rescue" has been attempted in an invertebrate. Remarkably, like mammals, maternal expression of certain MTX-resistant DHFRs in D. melanogaster can protect developing progeny from lethality and teratogenic effects in a dose-dependent manner. Thus, the similarity of teratogenic effects produced by MTX in mammals and Drosophila (Affleck et al., 2006b), the possibility of investigating alternative gene targets for MTX, and the ability to examine tissue-specific expression underscore Drosophila's potential as a model to investigate MTX-resistant DHFRs for gene therapy techniques and protection against MTX in a whole organism, while avoiding mammalian suffering and sacrifice.
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FUNDING |
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TOPABSTRACTINTRODUCTIONMETHODS AND MATERIALSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Natural Science and Engineering Research Council (NSERC) grant to V.K.W.; NSERC scholarship to J.G.A.
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ACKNOWLEDGMENTS |
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We thank all the previous students that tried to make the elusive "drug-resistant" flies over the years including Drs D. and S. Rancourt, Dr M. Tyshenko, Mr R. Hum, Dr. K A1-Batayneh and Ms K. Neumann. We also sincerely thank Dr L. Seroude for the pINDY5 vector and assistance with embryo injections.
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