Abstract
We use the Drosophila melanogaster larval hematopoietic system as an in vivo model for the genetic and functional genomic analysis of oncogenic cell overproliferation. Ras regulates cell proliferation and differentiation in multicellular eukaryotes. To further elucidate the role of activated Ras in cell overproliferation, we generated a collagen promoter-Gal4 strain to overexpress Ras(V12) in Drosophila hemocytes. Activated Ras causes a dramatic increase in the number of circulating larval hemocytes (blood cells), which is caused by cellular overproliferation. This phenotype is mediated by the Raf/MAPK pathway. The mutant hemocytes retain the ability to phagocytose bacteria as well as to differentiate into lamellocytes. Microarray analysis of hemocytes overexpressing Ras(V12) vs. Ras(+) identified 279 transcripts that are differentially expressed threefold or more in hemocytes expressing activated Ras. This work demonstrates that it will be feasible to combine genetic and functional genomic approaches in the Drosophila hematopoietic system to systematically identify oncogene-specific downstream targets.
Full Text
The Full Text of this article is available as a PDF (236.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Beaupre D. M., Kurzrock R. RAS and leukemia: from basic mechanisms to gene-directed therapy. J Clin Oncol. 1999 Mar;17(3):1071–1079. doi: 10.1200/JCO.1999.17.3.1071. [DOI] [PubMed] [Google Scholar]
- Bernardoni R., Vivancos V., Giangrande A. glide/gcm is expressed and required in the scavenger cell lineage. Dev Biol. 1997 Nov 1;191(1):118–130. doi: 10.1006/dbio.1997.8702. [DOI] [PubMed] [Google Scholar]
- Biggs W. H., 3rd, Zavitz K. H., Dickson B., van der Straten A., Brunner D., Hafen E., Zipursky S. L. The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway. EMBO J. 1994 Apr 1;13(7):1628–1635. doi: 10.1002/j.1460-2075.1994.tb06426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bos J. L. ras oncogenes in human cancer: a review. Cancer Res. 1989 Sep 1;49(17):4682–4689. [PubMed] [Google Scholar]
- Bradley P. L., Andrew D. J. ribbon encodes a novel BTB/POZ protein required for directed cell migration in Drosophila melanogaster. Development. 2001 Aug;128(15):3001–3015. doi: 10.1242/dev.128.15.3001. [DOI] [PubMed] [Google Scholar]
- Brand A. H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
- Chang H. C., Solomon N. M., Wassarman D. A., Karim F. D., Therrien M., Rubin G. M., Wolff T. phyllopod functions in the fate determination of a subset of photoreceptors in Drosophila. Cell. 1995 Feb 10;80(3):463–472. doi: 10.1016/0092-8674(95)90497-2. [DOI] [PubMed] [Google Scholar]
- Datar S. A., Jacobs H. W., de la Cruz A. F., Lehner C. F., Edgar B. A. The Drosophila cyclin D-Cdk4 complex promotes cellular growth. EMBO J. 2000 Sep 1;19(17):4543–4554. doi: 10.1093/emboj/19.17.4543. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Gregorio E., Spellman P. T., Rubin G. M., Lemaitre B. Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci U S A. 2001 Oct 16;98(22):12590–12595. doi: 10.1073/pnas.221458698. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dearolf C. R. Fruit fly "leukemia". Biochim Biophys Acta. 1998 Feb 20;1377(1):M13–M23. doi: 10.1016/s0304-419x(97)00031-0. [DOI] [PubMed] [Google Scholar]
- Dickson B. J., Domínguez M., van der Straten A., Hafen E. Control of Drosophila photoreceptor cell fates by phyllopod, a novel nuclear protein acting downstream of the Raf kinase. Cell. 1995 Feb 10;80(3):453–462. doi: 10.1016/0092-8674(95)90496-4. [DOI] [PubMed] [Google Scholar]
- Edgar B. A., Lehman D. A., O'Farrell P. H. Transcriptional regulation of string (cdc25): a link between developmental programming and the cell cycle. Development. 1994 Nov;120(11):3131–3143. doi: 10.1242/dev.120.11.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franc N. C., Dimarcq J. L., Lagueux M., Hoffmann J., Ezekowitz R. A. Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity. 1996 May;4(5):431–443. doi: 10.1016/s1074-7613(00)80410-0. [DOI] [PubMed] [Google Scholar]
- Gateff E. Malignant neoplasms of genetic origin in Drosophila melanogaster. Science. 1978 Jun 30;200(4349):1448–1459. doi: 10.1126/science.96525. [DOI] [PubMed] [Google Scholar]
- Greenwald I., Rubin G. M. Making a difference: the role of cell-cell interactions in establishing separate identities for equivalent cells. Cell. 1992 Jan 24;68(2):271–281. doi: 10.1016/0092-8674(92)90470-w. [DOI] [PubMed] [Google Scholar]
- Hafen E. Patterning by cell recruitment in the Drosophila eye. Curr Opin Genet Dev. 1991 Aug;1(2):268–274. doi: 10.1016/s0959-437x(05)80081-4. [DOI] [PubMed] [Google Scholar]
- Hanratty W. P., Ryerse J. S. A genetic melanotic neoplasm of Drosophila melanogaster. Dev Biol. 1981 Apr 30;83(2):238–249. doi: 10.1016/0012-1606(81)90470-x. [DOI] [PubMed] [Google Scholar]
- Harrison D. A., Binari R., Nahreini T. S., Gilman M., Perrimon N. Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J. 1995 Jun 15;14(12):2857–2865. doi: 10.1002/j.1460-2075.1995.tb07285.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hay B. A., Wolff T., Rubin G. M. Expression of baculovirus P35 prevents cell death in Drosophila. Development. 1994 Aug;120(8):2121–2129. doi: 10.1242/dev.120.8.2121. [DOI] [PubMed] [Google Scholar]
- Hendzel M. J., Wei Y., Mancini M. A., Van Hooser A., Ranalli T., Brinkley B. R., Bazett-Jones D. P., Allis C. D. Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma. 1997 Nov;106(6):348–360. doi: 10.1007/s004120050256. [DOI] [PubMed] [Google Scholar]
- Joneson T., White M. A., Wigler M. H., Bar-Sagi D. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS. Science. 1996 Feb 9;271(5250):810–812. doi: 10.1126/science.271.5250.810. [DOI] [PubMed] [Google Scholar]
- Jordan K. C., Clegg N. J., Blasi J. A., Morimoto A. M., Sen J., Stein D., McNeill H., Deng W. M., Tworoger M., Ruohola-Baker H. The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation. Nat Genet. 2000 Apr;24(4):429–433. doi: 10.1038/74294. [DOI] [PubMed] [Google Scholar]
- Khosravi-Far R., White M. A., Westwick J. K., Solski P. A., Chrzanowska-Wodnicka M., Van Aelst L., Wigler M. H., Der C. J. Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation. Mol Cell Biol. 1996 Jul;16(7):3923–3933. doi: 10.1128/mcb.16.7.3923. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Konrad L., Becker G., Schmidt A., Klöckner T., Kaufer-Stillger G., Dreschers S., Edström J. E., Gateff E. Cloning, structure, cellular localization, and possible function of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 of Drosophila melanogaster. Dev Biol. 1994 May;163(1):98–111. doi: 10.1006/dbio.1994.1126. [DOI] [PubMed] [Google Scholar]
- Lane M. E., Sauer K., Wallace K., Jan Y. N., Lehner C. F., Vaessin H. Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell. 1996 Dec 27;87(7):1225–1235. doi: 10.1016/s0092-8674(00)81818-8. [DOI] [PubMed] [Google Scholar]
- Lanot R., Zachary D., Holder F., Meister M. Postembryonic hematopoiesis in Drosophila. Dev Biol. 2001 Feb 15;230(2):243–257. doi: 10.1006/dbio.2000.0123. [DOI] [PubMed] [Google Scholar]
- Lebestky T., Chang T., Hartenstein V., Banerjee U. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science. 2000 Apr 7;288(5463):146–149. doi: 10.1126/science.288.5463.146. [DOI] [PubMed] [Google Scholar]
- Losi L., Roncucci L., di Gregorio C., de Leon M. P., Benhattar J. K-ras and p53 mutations in human colorectal aberrant crypt foci. J Pathol. 1996 Mar;178(3):259–263. doi: 10.1002/(SICI)1096-9896(199603)178:3<259::AID-PATH473>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
- Luo H., Rose P. E., Roberts T. M., Dearolf C. R. The Hopscotch Jak kinase requires the Raf pathway to promote blood cell activation and differentiation in Drosophila. Mol Genet Genomics. 2002 Feb 1;267(1):57–63. doi: 10.1007/s00438-001-0632-7. [DOI] [PubMed] [Google Scholar]
- Nelson R. E., Fessler L. I., Takagi Y., Blumberg B., Keene D. R., Olson P. F., Parker C. G., Fessler J. H. Peroxidasin: a novel enzyme-matrix protein of Drosophila development. EMBO J. 1994 Aug 1;13(15):3438–3447. doi: 10.1002/j.1460-2075.1994.tb06649.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neufeld T. P., de la Cruz A. F., Johnston L. A., Edgar B. A. Coordination of growth and cell division in the Drosophila wing. Cell. 1998 Jun 26;93(7):1183–1193. doi: 10.1016/s0092-8674(00)81462-2. [DOI] [PubMed] [Google Scholar]
- Neuman-Silberberg F. S., Schejter E., Hoffmann F. M., Shilo B. Z. The Drosophila ras oncogenes: structure and nucleotide sequence. Cell. 1984 Jul;37(3):1027–1033. doi: 10.1016/0092-8674(84)90437-9. [DOI] [PubMed] [Google Scholar]
- Neuman-Silberberg F. S., Schejter E., Hoffmann F. M., Shilo B. Z. The Drosophila ras oncogenes: structure and nucleotide sequence. Cell. 1984 Jul;37(3):1027–1033. doi: 10.1016/0092-8674(84)90437-9. [DOI] [PubMed] [Google Scholar]
- Prober D. A., Edgar B. A. Ras1 promotes cellular growth in the Drosophila wing. Cell. 2000 Feb 18;100(4):435–446. doi: 10.1016/s0092-8674(00)80679-0. [DOI] [PubMed] [Google Scholar]
- Qiu P., Pan P. C., Govind S. A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis. Development. 1998 May;125(10):1909–1920. doi: 10.1242/dev.125.10.1909. [DOI] [PubMed] [Google Scholar]
- Rehorn K. P., Thelen H., Michelson A. M., Reuter R. A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development. 1996 Dec;122(12):4023–4031. doi: 10.1242/dev.122.12.4023. [DOI] [PubMed] [Google Scholar]
- Rizki T. M., Rizki R. M., Bellotti R. A. Genetics of a Drosophila phenoloxidase. Mol Gen Genet. 1985;201(1):7–13. doi: 10.1007/BF00397978. [DOI] [PubMed] [Google Scholar]
- Russo J., Dupas S., Frey F., Carton Y., Brehelin M. Insect immunity: early events in the encapsulation process of parasitoid (Leptopilina boulardi) eggs in resistant and susceptible strains of Drosophila. Parasitology. 1996 Jan;112(Pt 1):135–142. doi: 10.1017/s0031182000065173. [DOI] [PubMed] [Google Scholar]
- Schulze A., Lehmann K., Jefferies H. B., McMahon M., Downward J. Analysis of the transcriptional program induced by Raf in epithelial cells. Genes Dev. 2001 Apr 15;15(8):981–994. doi: 10.1101/gad.191101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spruck C., Strohmaier H., Watson M., Smith A. P., Ryan A., Krek T. W., Reed S. I. A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1. Mol Cell. 2001 Mar;7(3):639–650. doi: 10.1016/s1097-2765(01)00210-6. [DOI] [PubMed] [Google Scholar]
- Tang A. H., Neufeld T. P., Kwan E., Rubin G. M. PHYL acts to down-regulate TTK88, a transcriptional repressor of neuronal cell fates, by a SINA-dependent mechanism. Cell. 1997 Aug 8;90(3):459–467. doi: 10.1016/s0092-8674(00)80506-1. [DOI] [PubMed] [Google Scholar]
- Thummel C. S., Boulet A. M., Lipshitz H. D. Vectors for Drosophila P-element-mediated transformation and tissue culture transfection. Gene. 1988 Dec 30;74(2):445–456. doi: 10.1016/0378-1119(88)90177-1. [DOI] [PubMed] [Google Scholar]
- Van Vactor D. L., Jr, Cagan R. L., Krämer H., Zipursky S. L. Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell. Cell. 1991 Dec 20;67(6):1145–1155. doi: 10.1016/0092-8674(91)90291-6. [DOI] [PubMed] [Google Scholar]
- White M. A., Nicolette C., Minden A., Polverino A., Van Aelst L., Karin M., Wigler M. H. Multiple Ras functions can contribute to mammalian cell transformation. Cell. 1995 Feb 24;80(4):533–541. doi: 10.1016/0092-8674(95)90507-3. [DOI] [PubMed] [Google Scholar]
- Yasothornsrikul S., Davis W. J., Cramer G., Kimbrell D. A., Dearolf C. R. viking: identification and characterization of a second type IV collagen in Drosophila. Gene. 1997 Oct 1;198(1-2):17–25. doi: 10.1016/s0378-1119(97)00274-6. [DOI] [PubMed] [Google Scholar]
- Yasothornsrikul S., Davis W. J., Cramer G., Kimbrell D. A., Dearolf C. R. viking: identification and characterization of a second type IV collagen in Drosophila. Gene. 1997 Oct 1;198(1-2):17–25. doi: 10.1016/s0378-1119(97)00274-6. [DOI] [PubMed] [Google Scholar]
- Zinyk D. L., McGonnigal B. G., Dearolf C. R. Drosophila awdK-pn, a homologue of the metastasis suppressor gene nm23, suppresses the Tum-1 haematopoietic oncogene. Nat Genet. 1993 Jun;4(2):195–201. doi: 10.1038/ng0693-195. [DOI] [PubMed] [Google Scholar]
- de Nooij J. C., Hariharan I. K. Uncoupling cell fate determination from patterned cell division in the Drosophila eye. Science. 1995 Nov 10;270(5238):983–985. doi: 10.1126/science.270.5238.983. [DOI] [PubMed] [Google Scholar]
- de Nooij J. C., Letendre M. A., Hariharan I. K. A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell. 1996 Dec 27;87(7):1237–1247. doi: 10.1016/s0092-8674(00)81819-x. [DOI] [PubMed] [Google Scholar]
