One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53-activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS-p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53-induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2, Rga, crol, and Spt5). In the other 3 (Orct2, Polr2M, and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death.

Aktogen Hungary Ltd 6726 Szeged Hungary

Bioinformatics Laboratory Core Facility HUN REN Biological Research Centre 6726 Szeged Hungary

Biology Centre Czech Academy of Sciences 37005 České Budějovice Czech Republic

Brain Research Institute University of Zurich 8057 Zurich Switzerland

Cellular Imaging Laboratory Core Facility HUN REN Biological Research Centre 6726 Szeged Hungary

Centre for Agricultural Sciences Plant Protection Institute 1022 Budapest Hungary

Department of Biochemistry and Medical Chemistry Medical School University of Pécs 7624 Pécs Hungary

Department of Developmental Genetics German Cancer Research Centre 69120 Heidelberg Germany

Institute of Biochemistry HUN REN Biological Research Centre 6726 Szeged Hungary

Institute of Genetics HUN REN Biological Research Centre 6726 Szeged Hungary

Laboratory of Functional Genomics Core Facility HUN REN Biological Research Centre 6726 Szeged Hungary

Zobrazit více v PubMed

Abbas H, Derkaoui DK, Jeammet L, Adicéam E, Tiollier J, Sicard H, Braun T, Poyet JL. 2024. Apoptosis inhibitor 5: a multifaceted regulator of cell fate. Biomolecules. 14(1):136. doi:10.3390/biom14010136. PubMed DOI PMC

Alvarado-Ortiz E, de la Cruz-López KG, Becerril-Rico J, Sarabia-Sánchez MA, Ortiz-Sánchez E, García-Carrancá A. 2020. Mutant p53 gain-of-function: role in cancer development, progression, and therapeutic approaches. Front Cell Dev Biol. 8:607670. doi:10.3389/fcell.2020.607670. PubMed DOI PMC

Anbarasan T, Bourdon J-C. 2019. The emerging landscape of p53 isoforms in physiology, cancer and degenerative diseases. Int J Mol Sci. 20(24):6257. doi:10.3390/ijms20246257. PubMed DOI PMC

Aubrey BJ, Janic A, Chen Y, Chang C, Lieschke EC, Diepstraten ST, Kueh AJ, Bernardini JP, Dewson G, O'Reilly LA, et al. . 2018a. Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development. Genes Dev. 32(21–22):1420–1429. doi:10.1101/gad.314286.118. PubMed DOI PMC

Aubrey BJ, Kelly GL, Janic A, Herold MJ, Strasser A. 2018b. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell Death Differ. 25(1):104–113. doi:10.1038/cdd.2017.169. PubMed DOI PMC

Ben-Shahar Y, Nannapaneni K, Casavant TL, Scheetz TE, Welsh MJ. 2007. Eukaryotic operon-like transcription of functionally related genes in Drosophila. Proc Natl Acad Sci U S A. 104(1):222–227. doi:10.1073/pnas.0609683104. PubMed DOI PMC

Bertheloot D, Latz E, Franklin BS. 2021. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell Mol Immunol. 18(5):1106–1121. doi:10.1038/s41423-020-00630-3. PubMed DOI PMC

Boettcher S, Miller PG, Sharma R, McConkey M, Leventhal M, Krivtsov AV, Giacomelli AO, Wong W, Kim J, Chao S, et al. . 2019. A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies. Science. 365(6453):599–604. doi:10.1126/science.aax3649. PubMed DOI PMC

Boutanaev AM, Kalmykova AI, Shevelyov YY, Nurminsky DI. 2002. Large clusters of co-expressed genes in the Drosophila genome. Nature. 420(6916):666–669. doi:10.1038/nature01216. PubMed DOI

Brachmann CB, Jassim OW, Wachsmuth BD, Cagan RL. 2000. The Drosophila bcl-2 family member dBorg-1 functions in the apoptotic response to UV-irradiation. Curr Biol. 10(9):547–550. doi:10.1016/s0960-9822(00)00474-7. PubMed DOI

Brand AH, Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Dev Camb Engl. 118:401–415. doi:10.1242/dev.118.2.401. PubMed DOI

Bredesen DE, Rao RV, Mehlen P. 2006. Cell death in the nervous system. Nature. 443(7113):796–802. doi:10.1038/nature05293. PubMed DOI PMC

Brodsky MH, Nordstrom W, Tsang G, Kwan E, Rubin GM, Abrams JM. 2000. Drosophila p53 binds a damage response element at the reaper locus. Cell. 101(1):103–113. doi:10.1016/S0092-8674(00)80627-3. PubMed DOI

Cetraro P, Plaza-Diaz J, MacKenzie A, Abadía-Molina F. 2022. A review of the current impact of inhibitors of apoptosis proteins and their repression in cancer. Cancers (Basel). 14(7):1671. doi:10.3390/cancers14071671. PubMed DOI PMC

Chakraborty R, Li Y, Zhou L, Golic KG. 2015. Corp regulates P53 in Drosophila melanogaster via a negative feedback loop. PLoS Genet. 11(7):e1005400. doi:10.1371/journal.pgen.1005400. PubMed DOI PMC

Chakravarti A, Thirimanne HN, Brown S, Calvi BR. 2022. Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control. eLife. 11:e61389. doi:10.7554/eLife.61389. PubMed DOI PMC

Cho YC, Park JE, Park BC, Kim JH, Jeong DG, Park SG, Cho S. 2015. Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1. Cell Death Differ. 22(10):1605–1617. doi:10.1038/cdd.2015.2. PubMed DOI PMC

Colussi PA, Quinn LM, Huang DC, Coombe M, Read SH, Richardson H, Kumar S. 2000. Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery. J Cell Biol. 148(4):703–714. doi:10.1083/jcb.148.4.703. PubMed DOI PMC

D’Brot A, Kurtz P, Regan E, Jakubowski B, Abrams JM. 2017. A platform for interrogating cancer-associated p53 alleles. Oncogene. 36(2):286–291. doi:10.1038/onc.2016.48. PubMed DOI PMC

De S, Babu MM. 2010. Genomic neighbourhood and the regulation of gene expression. Curr Opin Cell Biol. 22(3):326–333. doi:10.1016/j.ceb.2010.04.004. PubMed DOI

Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW, Vogelstein B. 1993. WAF1, a potential mediator of p53 tumor suppression. Cell. 75(4):817–825. doi:10.1016/0092-8674(93)90500-p. PubMed DOI

Dietzl G, Chen D, Schnorrer F, Su KC, Barinova Y, Fellner M, Gasser B, Kinsey K, Oppel S, Scheiblauer S, et al. . 2007. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature. 448(7150):151–156. doi:10.1038/nature05954. PubMed DOI

Domingues C, Ryoo HD. 2012. Drosophila BRUCE inhibits apoptosis through non-lysine ubiquitination of the IAP-antagonist REAPER. Cell Death Differ. 19(3):470–477. doi:10.1038/cdd.2011.116. PubMed DOI PMC

Engeland K. 2018. Cell cycle arrest through indirect transcriptional repression by p53: i have a DREAM. Cell Death Differ. 25(1):114–132. doi:10.1038/cdd.2017.172. PubMed DOI PMC

Engeland K. 2022. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ. 29(5):946–960. doi:10.1038/s41418-022-00988-z. PubMed DOI PMC

Fairbrother WJ, Leverson JD, Sampath D, Souers AJ. 2019. Discovery and development of venetoclax, a selective antagonist of BCL-2. In: Successful Drug Discovery. Hoboken, NJ: John Wiley & Sons, Ltd. p. 225–245.

Fan Y, Lee TV, Xu D, Chen Z, Lamblin AF, Steller H, Bergmann A. 2010. Dual roles of Drosophila p53 in cell death and cell differentiation. Cell Death Differ. 17(6):912–921. doi:10.1038/cdd.2009.182. PubMed DOI PMC

Freeman M. 1996. Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell. 87(4):651–660. doi:10.1016/s0092-8674(00)81385-9. PubMed DOI

Fuhrmann G, Leisser C, Rosenberger G, Grusch M, Huettenbrenner S, Halama T, Mosberger I, Sasgary S, Cerni C, Krupitza G. 2001. Cdc25A phosphatase suppresses apoptosis induced by serum deprivation. Oncogene. 20(33):4542–4553. doi:10.1038/sj.onc.1204499. PubMed DOI

Hay BA, Wassarman DA, Rubin GM. 1995. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell. 83(7):1253–1262. doi:10.1016/0092-8674(95)90150-7. PubMed DOI

Hou H, Sun D, Zhang X. 2019. The role of MDM2 amplification and overexpression in therapeutic resistance of malignant tumors. Cancer Cell Int. 19(1):216. doi:10.1186/s12935-019-0937-4. PubMed DOI PMC

Im J-Y, Kang M-J, Kim B-K, Won M. 2023. DDIAS, DNA damage-induced apoptosis suppressor, is a potential therapeutic target in cancer. Exp Mol Med. 55(5):879–885. doi:10.1038/s12276-023-00974-6. PubMed DOI PMC

Jin S, Martinek S, Joo WS, Wortman JR, Mirkovic N, Sali A, Yandell MD, Pavletich NP, Young MW, Levine AJ. 2000. Identification and characterization of a p53 homologue in Drosophila melanogaster. Proc Natl Acad Sci U S A. 97(13):7301–7306. doi:10.1073/pnas.97.13.7301. PubMed DOI PMC

Kaloni D, Diepstraten ST, Strasser A, Kelly GL. 2023. BCL-2 protein family: attractive targets for cancer therapy. Apoptosis. 28(1–2):20–38. doi:10.1007/s10495-022-01780-7. PubMed DOI PMC

Kim H, Lee JM, Lee G, Bhin J, Oh SK, Kim K, Pyo KE, Lee JS, Yim HY, Kim KI, et al. . 2011. DNA damage-induced RORα is crucial for p53 stabilization and increased apoptosis. Mol. Cell. 44(5):797–810. doi:10.1016/j.molcel.2011.09.023. PubMed DOI

Kramer JM, Staveley BE. 2003. GAL4 causes developmental defects and apoptosis when expressed in the developing eye of Drosophila melanogaster. Genet Mol Res. 2:43–47. PubMed

Kurenova E, Xu LH, Yang X, Baldwin AS, Craven RJ, Hanks SK, Liu ZG, Cance WG. 2004. Focal adhesion kinase suppresses apoptosis by binding to the death domain of receptor-interacting protein. Mol Cell Biol. 24(10):4361–4371. doi:10.1128/MCB.24.10.4361-4371.2004. PubMed DOI PMC

Kylsten P, Saint R. 1997. Imaginal tissues of Drosophila melanogaster exhibit different modes of cell proliferation control. Dev Biol. 192(2):509–522. doi:10.1006/dbio.1997.8770. PubMed DOI

Kyriacou CP. 2000. Drosophila protocols edited by William Sullivan, Michael Ashburner and R. Scott Hawley. Trends Genet. 16(11):524. doi:10.1016/S0168-9525(00)02094-1. DOI

Lercher MJ, Urrutia AO, Hurst LD. 2002. Clustering of housekeeping genes provides a unified model of gene order in the human genome. Nat Genet. 31(2):180–183. doi:10.1038/ng887. PubMed DOI

Li W-Z, Li S-L, Zheng HY, Zhang S-P, Xue L. 2012. A broad expression profile of the GMR-GAL4 driver in Drosophila melanogaster. Genet Mol Res. 11(3):1997–2002. doi:10.4238/2012.August.6.4. PubMed DOI

Liang J, Niu Z, Zhang B, Yu X, Zheng Y, Wang C, Ren H, Wang M, Ruan B, Qin H, et al. . 2021. p53-dependent elimination of aneuploid mitotic offspring by entosis. Cell Death Differ. 28(2):799–813. doi:10.1038/s41418-020-00645-3. PubMed DOI PMC

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 25(4):402–408. doi:10.1006/meth.2001.1262. PubMed DOI

Lukacsovich T, Asztalos Z, Awano W, Baba K, Kondo S, Niwa S, Yamamoto D. 2001. Dual-tagging gene trap of novel genes in Drosophila melanogaster. Genetics. 157(2):727–742. doi:10.1093/genetics/157.2.727. PubMed DOI PMC

Mehta S, Campbell H, Drummond CJ, Li K, Murray K, Slatter T, Bourdon JC, Braithwaite AW. 2021. Adaptive homeostasis and the p53 isoform network. EMBO Rep. 22(12):e53085. doi:10.15252/embr.202153085. PubMed DOI PMC

Michalak P. 2008. Coexpression, coregulation, and cofunctionality of neighboring genes in eukaryotic genomes. Genomics. 91(3):243–248. doi:10.1016/j.ygeno.2007.11.002. PubMed DOI

Mihelčić M, Šmuc T, Supek F. 2019. Patterns of diverse gene functions in genomic neighborhoods predict gene function and phenotype. Sci Rep. 9(1):19537. doi:10.1038/s41598-019-55984-0. PubMed DOI PMC

Morris EJ, Michaud WA, Ji JY, Moon NS, Rocco JW, Dyson NJ. 2006. Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo. PLoS Genet. 2(11):e196. doi:10.1371/journal.pgen.0020196. PubMed DOI PMC

Neufeld TP, de la Cruz AF, Johnston LA, Edgar BA. 1998. Coordination of growth and cell division in the Drosophila wing. Cell. 93(7):1183–1193. doi:10.1016/s0092-8674(00)81462-2. PubMed DOI

Ollmann M, Young LM, Di Como CJ, Karim F, Belvin M, Robertson S, Whittaker K, Demsky M, Fisher WW, Buchman A, et al. . 2000. Drosophila p53 is a structural and functional homolog of the tumor suppressor p53. Cell. 101(1):91–101. doi:10.1016/S0092-8674(00)80626-1. PubMed DOI

Öztürk-Çolak A, Marygold SJ, Antonazzo G, Attrill H, Goutte-Gattat D, Jenkins VK, Matthews BB, Millburn G, Dos Santos G, Tabone CJ. 2024. FlyBase: updates to the Drosophila genes and genomes database. Genetics. 227:iyad211. doi:10.1093/genetics/iyad211. PubMed DOI PMC

Pakos-Zebrucka K, Koryga I, Mnich K, Ljujic M, Samali A, Gorman AM. 2016. The integrated stress response. EMBO Rep. 17(10):1374–1395. doi:10.15252/embr.201642195. PubMed DOI PMC

Pardi N, Vámos E, Ujfaludi Z, Komonyi O, Bodai L, Boros IM. 2011. In vivo effects of abolishing the single canonical sumoylation site in the C-terminal region of Drosophila p53. Acta Biol Hung. 62(4):397–412. doi:10.1556/ABiol.62.2011.4.6. PubMed DOI

Peng F, Liao M, Qin R, Zhu S, Peng C, Fu L, Chen Y, Han B. 2022. Regulated cell death (RCD) in cancer: key pathways and targeted therapies. Signal Transduct Target Ther. 7(1):286. doi:10.1038/s41392-022-01110-y. PubMed DOI PMC

Quinn L, Coombe M, Mills K, Daish T, Colussi P, Kumar S, Richardson H. 2003. Buffy, a Drosophila Bcl-2 protein, has anti-apoptotic and cell cycle inhibitory functions. EMBO J. 22(14):3568–3579. doi:10.1093/emboj/cdg355. PubMed DOI PMC

Ray M, Lakhotia SC. 2015. The commonly used eye-specific sev-GAL4 and GMR-GAL4 drivers in Drosophila melanogaster are expressed in tissues other than eyes also. J Genet. 94(3):407–416. doi:10.1007/s12041-015-0535-8. PubMed DOI

Rizzotto D, Englmaier L, Villunger A. 2021. At a crossroads to cancer: how p53-induced cell fate decisions secure genome integrity. Int J Mol Sci. 22(19):10883. doi:10.3390/ijms221910883. PubMed DOI PMC

Robertson HM, Preston CR, Phillis RW, Johnson-Schlitz DM, Benz WK, Engels WR. 1988. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 118(3):461–470. doi:10.1093/genetics/118.3.461. PubMed DOI PMC

Roman G, Davis RL. 2002. Conditional expression of UAS-transgenes in the adult eye with a new gene-switch vector system. Genes (Basel). 34(1–2):127–131. doi:10.1002/gene.10133. PubMed DOI

Rørth P. 1996. A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci U S A. 93(22):12418–12422. doi:10.1073/pnas.93.22.12418. PubMed DOI PMC

Ruiz-Losada M, González R, Peropadre A, Gil-Gálvez A, Tena JJ, Baonza A, Estella C. 2022. Coordination between cell proliferation and apoptosis after DNA damage in Drosophila. Cell Death Differ. 29(4):832–845. doi:10.1038/s41418-021-00898-6. PubMed DOI PMC

Sammons MA, Nguyen T-AT, McDade SS, Fischer M. 2020. Tumor suppressor p53: from engaging DNA to target gene regulation. Nucleic Acids Res. 48(16):8848–8869. doi:10.1093/nar/gkaa666. PubMed DOI PMC

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. . 2012. Fiji: an open-source platform for biological-image analysis. Nat Methods. 9(7):676–682. doi:10.1038/nmeth.2019. PubMed DOI PMC

Shilova VY, Garbuz DG, Myasyankina EN, Chen B, Evgen'Ev MB, Feder ME, Zatsepina OG. 2006. Remarkable site specificity of local transposition into the Hsp70 promoter of Drosophila melanogaster. Genetics. 173(2):809–820. doi:10.1534/genetics.105.053959. PubMed DOI PMC

Singh R, Letai A, Sarosiek K. 2019. Regulation of apoptosis in health and disease: the balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol. 20(3):175–193. doi:10.1038/s41580-018-0089-8. PubMed DOI PMC

Sonoda Y, Matsumoto Y, Funakoshi M, Yamamoto D, Hanks SK, Kasahara T. 2000. Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60. J Biol Chem. 275(21):16309–16315. doi:10.1074/jbc.275.21.16309. PubMed DOI

Spellman PT, Rubin GM. 2002. Evidence for large domains of similarly expressed genes in the Drosophila genome. J Biol. 1(1):5. doi:10.1186/1475-4924-1-5. PubMed DOI PMC

Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M. 2016. The GeneCards suite: from gene data mining to disease genome sequence analyses. Curr Protoc Bioinforma. 54:1.30.1–1.30.33. doi:10.1002/cpbi.5. PubMed DOI

Stolc V, Gauhar Z, Mason C, Halasz G, Van Batenburg MF, Rifkin SA, Hua S, Herreman T, Tongprasit W, Barbano PE, et al. . 2004. A gene expression map for the euchromatic genome of Drosophila melanogaster. Science. 306(5696):655–660. doi:10.1126/science.1101312. PubMed DOI

Toba G, Ohsako T, Miyata N, Ohtsuka T, Seong KH, Aigaki T. 1999. The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster. Genetics. 151(2):725–737. doi:10.1093/genetics/151.2.725. PubMed DOI PMC

Voss AK, Strasser A. 2020. The essentials of developmental apoptosis. F1000Res. 9:148. doi:10.12688/f1000research.21571.1. PubMed DOI PMC

Wang Z, Burigotto M, Ghetti S, Vaillant F, Tan T, Capaldo BD, Palmieri M, Hirokawa Y, Tai L, Simpson DS, et al. . 2024. Loss-of-function but not gain-of-function properties of mutant TP53 are critical for the proliferation, survival, and metastasis of a broad range of cancer cells. Cancer Discov. 14(2):362–379. doi:10.1158/2159-8290.CD-23-0402. PubMed DOI PMC

Yan HF, Zou T, Tuo QZ, Xu S, Li H, Belaidi AA, Lei P. 2021. Ferroptosis: mechanisms and links with diseases. Signal Transduct Target Ther. 6:49. doi:10.1038/s41392-020-00428-9. PubMed DOI PMC

Yang Z, Edenberg HJ, Davis RL. 2005. Isolation of mRNA from specific tissues of Drosophila by mRNA tagging. Nucleic Acids Res. 33(17):e148. doi:10.1093/nar/gni149. PubMed DOI PMC

Yu P, Zhang X, Liu N, Tang L, Peng C, Chen X. 2021. Pyroptosis: mechanisms and diseases. Signal Transduct Target Ther. 6(1):128. doi:10.1038/s41392-021-00507-5. PubMed DOI PMC

Zhang B, Mehrotra S, Ng WL, Calvi BR. 2014. Low levels of p53 protein and chromatin silencing of p53 target genes repress apoptosis in Drosophila endocycling cells. PLoS Genet. 10(9):e1004581. doi:10.1371/journal.pgen.1004581. PubMed DOI PMC

Zhang B, Rotelli M, Dixon M, Calvi BR. 2015. The function of Drosophila p53 isoforms in apoptosis. Cell Death Differ. 22(12):2058–2067. doi:10.1038/cdd.2015.40. PubMed DOI PMC

Zhou L. 2019. P53 and apoptosis in the Drosophila model. Adv Exp Med Biol. 1167:105–112. doi:10.1007/978-3-030-23629-8_6. PubMed DOI