Exploring Protection from Methotrexate-Induced Teratogenicity in Flies
Division of Genetic Disorders, Wadsworth Center, New York State Department of Health, Albany, New York 12208; and Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, New York 12201
1 For correspondence via fax: (518) 474-3181. E-mail: wwolfgan{at}wadsworth.org.
Received July 26, 2007; accepted July 27, 2007
Key Words: methotrexate; Drosophila; developmental/teratology; reproductive and developmental toxicology.
For virtually all drug therapies, there exists a balance between efficacy and toxicity. This is particularly true in the treatment of many forms of cancer; such treatment often entails relatively nonspecific targeting of rapidly dividing cells by a variety of toxic agents. Clearly, the therapeutic efficacy of existing treatments would be improved if the toxicity of these agents could be reduced.
One such agent, methotrexate (8-amino-10-methyl-pteroyglutamic acid; MTX), has been used since the 1950s to treat childhood leukemia. The long-term value of this drug is apparent from the continued clinical reliance on it: MTX is commonly used to treat a variety of cancers including leukemia, osteosarcoma, breast cancer, and head and neck cancers. Its use has expanded to include a number of other diseases, including rheumatoid arthritis, psoriasis, Crohn's disease, asthma, and lupus. It is also utilized to terminate ectopic pregnancies and can be used to induce abortion (McGuire, 2003
). Thus, rather than being superseded by newer drugs, MTX continues to find new applications (Nguyen et al., 2002).
MTX is a synthetic analogue of dihydrofolate (DHF) and acts as a potent inhibitor of the housekeeping enzyme dihydrofolate reductase (DHFR). Inhibition of DHFR blocks the conversion of DHF to tetrahydrofolate (THF). THF in turn is an essential cofactor in the biosynthesis of purines, thymidylate, and some amino acids. Through depletion of the pool of THF, MTX disrupts DNA synthesis and causes rapidly dividing cells to arrest and die. Its toxicity toward rapidly dividing cells forms the basis of its therapeutic effect in cancer.
Unfortunately, because of its effect on rapidly dividing cells, MTX has also proven to be a potent teratogen in humans as well as in animal models. Birth defects in children born to women who have been treated with MTX for cancer or other conditions during pregnancy have been extensively characterized. These include skeletal defects, low birth weight, and a wide range of developmental abnormalities (Lloyd et al., 1999). In efforts to reduce these toxic affects and improve efficacy, animal models in mice, rats, and rabbits have been developed, for the purpose of determining the mechanism of teratogenicity and attempting to develop protective strategies. Such strategies include the use of structural analogues of THF to partially alleviate pathology in a rabbit model and in humans, at the clinical level (DeSesso and Goeringer, 1991; McGuire, 2003). More recently, applying molecular approaches combined with gene therapy, transgenic hematopoietic cell populations have been created; these carry a mutated form of DHFR that is resistant to MTX inhibition (Budak-Alpdogan et al., 2005; Sorrentino, 2002). When transplanted back into host animals, cells harboring the MTX-resistant form of DHFR could survive longer and in the presence of higher concentrations of MTX, than could their untransformed progenitors. However, none of the models so far developed combines the ability to characterize MTX-induced teratogenesis at the molecular level with the capability of identifying MTX-resistant DHFR variants that block teratogenesis in an intact animal. The creation of a Drosophila model of MTX-induced teratogenesis, by Virginia Walker's lab, fills this void and should enable us to gain a more complete understanding of the role of MTX and DHFR in teratogenesis.
The development of human disease models in Drosophila is possible because of the conservation of both disease genes and biochemical pathways between flies and man (Chien et al., 2002). Fly models of neurodegenerative diseases, cancer, inborn errors of metabolism, and developmental and hematopoietic pathologies all take advantage of the insect's facile genetics, to characterize disease pathways and to develop therapeutic interventions in an intact, functioning organism. The short generation time and low cost of rearing permit genetic experimentation and analyses that are not possible in mammals. Crucially, novel findings in flies have frequently been the basis for new lines of investigation in vertebrate systems.
Creation of the Walker lab's MTX-induced teratogenic Drosophila model started with the finding that upon feeding of MTX to adult flies, females over a 2- to 3-day period ceased to lay eggs, while their viability was unaffected (Affleck et al., 2006b). When the ovaries of these females were examined, they were found to be morphologically abnormal, as were the few eggs produced. In addition, the few progeny that survived exhibited a variety of pathologies, including melanotic tumors and exoskeletal defects. Importantly, these abnormalities were associated with populations of cells that were rapidly dividing. Further, microarray analysis pointed to altered expression of genes involved in regulation of the cell cycle, DNA damage repair, stress response, and embryonic development.
Thus, armed with a phenotype that had many parallels with MTX toxicity seen in humans, Walker and her colleagues wished to determine whether they could isolate Drosophila variants of DHFR that were resistant to MTX inhibition (Affleck et al., 2006a). They used three strategies in their isolation attempts. (1) Flies were reared over a period of 8 years on food containing MTX to select for resistant lines. This yielded K31P and Q134K DHFR variants. (2) Selection for MTX resistance in the S3 Drosophila cell line yielded a L30Q variant. (3) In silico analysis yielded a L22R variant. These four variants, plus wild-type Drosophila DHFR, were transfected individually into Chinese hamster ovary (CHO) cells that contained no detectable endogenous DHFR. Importantly, wild-type Drosophila DHFR could substitute for the mammalian version of the gene in the CHO cells. Of the four variants tested, only L30Q and L22R demonstrated MTX resistance.
Building on the two earlier studies, the current work by the lab tests the hypothesis that expression of MTX-resistant DHFR in female flies can suppress MTX-induced teratogenesis observed in the eggs and progeny (Affleck and Walker, 2007). The testing entails transformation of flies with the wild-type, L30Q, or L22R DHFR gene. Each of these transgenes has been engineered to be downstream of the yeast Gal4-binding sequence (upstream activating sequence), and each is missing its endogenous promoter. Thus, each is silent unless yeast Gal4 protein, normally absent in flies, is supplied. When DHFR transgenic flies are crossed to flies expressing Gal4 protein, the progeny expresses DHFR in the same pattern as the Gal4 protein. Since labs and stock centers maintain many different lines of flies that have Gal4 protein expressed in a variety of known patterns, it is straightforward to control pattern of DHFR production by crossing to different Gal4 lines.
Female flies ubiquitously expressing the wild-type or resistant forms of DHFR were produced and exposed to 1µM MTX. After 2–3 days, only the flies harboring the L30Q variant continued to lay eggs at a normal rate, demonstrating that the L30Q variant, but not the wild-type or L22R form, is resistant to the teratogenic effects of MTX on the fly. Furthermore, the ovaries and eggs of the L30Q flies were morphologically normal. Similar results were obtained when two other Gal4 drivers were each used to restrict L30Q DHFR expression to the ovaries. These findings indicate that MTX-induced teratogenesis is mediated at least in part by DHFR and that the ovaries are the site of action. Microarray analysis of ovaries demonstrated that the altered transcript abundances associated with MTX treatment in the previous studies were largely reproduced in flies transformed with either wild-type or the L22R variant DHFR. Strikingly, for L30Q transformed flies, transcript levels were more like untreated flies.
The Drosophila model developed by the Walker group will continue to be valuable in the effort to understand mechanisms of and derive protective solutions for MTX-induced teratogenesis. The fly model has a number of advantages over the limited number of available mammalian models. (1) Variant forms of DHFR can be assessed rapidly and inexpensively for resistance to MTX teratogenesis in an intact animal. The capability to screen many variants is critical given that variant forms, which show exciting potential in tissue culture studies, may be ineffective when tested in the intact organism (i.e., L22R [Affleck et al., 2006b]). (2) Molecular mechanisms of MTX toxicity and variant DHFR rescue can now be examined in depth, via a variety of genetic strategies in Drosophila that can query the entire genome for other genes that interact functionally to enhance or suppress teratogenesis. Identification of interacting loci can in turn lead to the identification of unexpected pathways susceptible to modification through drug and/or gene therapy strategies to further block MTX toxicity. These functional genome-wide screens are far more difficult, costly, and problematic to perform in vertebrate systems. (3) The model will also aid in distinguishing DHFR-dependent pathologies resulting from MTX exposure from DHFR-independent ones, based on the pathological response in the presence MTX-resistant variants. Combining therapies that affect two distinct pathways could confer a synergistic benefit, further reducing both the toxic and teratogenic properties of MTX. Information acquired through the comprehensive analysis of teratogenic mechanisms in flies can be used to develop and test novel strategies to reduce MTX toxicity first in preclinical mammalian studies and finally in the clinic itself.
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