Skip to main content
SpringerLink
Log in

Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy

  • Protocol
  • First Online:
Retinoid and Rexinoid Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2019))

Abstract

Acute myeloid leukemia (AML) is characterized by the accumulation of immature white blood cell precursors in the bone marrow and peripheral circulation. In essence, leukemic cells fail to differentiate and are stalled at a particular step of hematopoietic maturation and are unable to complete their development into functional blood cells with a finite life cycle. They are thus said to possess a “differentiation block.” Pharmacological override of this block is one attractive avenue of therapy, termed “differentiation therapy.” The most successful example of this therapeutic strategy is the use of the physiologic retinoid all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). In this chapter, we will outline the methods used to characterize the mechanisms mobilized by retinoid signaling and will use the activation of a key regulator of autophagy, ATG7, as an example of the functional characterization of a retinoid regulated gene during differentiation. We will discuss how lentiviral delivery of shRNA constructs into cultured APL cells, such as NB4, can be used to functionally deplete key proteins. We will also describe how the effect of protein knockdown on ATRA-induced differentiation and autophagy can be assessed using quantitative PCR, Western blotting, and flow cytometry.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 149.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Gudas LJ (1994) Retinoids and vertebrate development. J Biol Chem 269:15399–15402

    CAS  PubMed  Google Scholar 

  2. Laursen KB, Wong PM, Gudas LJ (2012) Epigenetic regulation by RARalpha maintains ligand-independent transcriptional activity. Nucleic Acids Res 40:102–115

    Article  CAS  Google Scholar 

  3. Kashyap V, Gudas LJ (2010) Epigenetic regulatory mechanisms distinguish retinoic acid-mediated transcriptional responses in stem cells and fibroblasts. J Biol Chem 285:14534–14548

    Article  CAS  Google Scholar 

  4. Kashyap V, Gudas LJ, Brenet F, Funk P, Viale A, Scandura JM (2011) Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid. J Biol Chem 286:3250–3260

    Article  CAS  Google Scholar 

  5. Purton LE, Bernstein ID, Collins SJ (2000) All-trans retinoic acid enhances the long-term repopulating activity of cultured hematopoietic stem cells. Blood 95:470–477

    CAS  PubMed  Google Scholar 

  6. Purton LE, Dworkin S, Olsen GH, Walkley CR, Fabb SA, Collins SJ, Chambon P (2006) RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation. J Exp Med 203:1283–1293

    Article  CAS  Google Scholar 

  7. Ghatpande S, Ghatpande A, Sher J, Zile MH, Evans T (2002) Retinoid signaling regulates primitive (yolk sac) hematopoiesis. Blood 99:2379–2386

    Article  CAS  Google Scholar 

  8. Ross AC (2012) Vitamin A and retinoic acid in T cell-related immunity. Am J Clin Nutr 96:1166S–1172S

    Article  CAS  Google Scholar 

  9. Mucida D, Park Y, Kim G, Turovskaya O, Scott I, Kronenberg M, Cheroutre H (2007) Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid. Science 317:256–260

    Article  CAS  Google Scholar 

  10. Caceres-Cortes JR (2013) Blastic leukaemias (AML): a biologist's view. Cell Biochem Biophys 66:13–22

    Article  CAS  Google Scholar 

  11. Lengfelder E, Hofmann WK, Nolte F (2013) Management of elderly patients with acute promyelocytic leukemia: progress and problems. Ann Hematol 92:1181–1188

    Article  Google Scholar 

  12. Grignani F, De Matteis S, Nervi C, Tomassoni L, Gelmetti V, Cioce M, Fanelli M, Ruthardt M, Ferrara FF, Zamir I, Seiser C, Grignani F, Lazar MA, Minucci S, Pelicci PG (1998) Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 391:815–818

    Article  CAS  Google Scholar 

  13. Di Croce L, Raker VA, Corsaro M, Fazi F, Fanelli M, Faretta M, Fuks F, Lo Coco F, Kouzarides T, Nervi C, Minucci S, Pelicci PG (2002) Methyltransferase recruitment and DNA hypermethylation of target promoters by an oncogenic transcription factor. Science 295:1079–1082

    Article  Google Scholar 

  14. Kayser S, Krzykalla J, Elliott MA, Norsworthy K, Gonzales P, Hills RK, Baer MR, Racil Z, Mayer J, Novak J, Zak P, Szotkowski T, Grimwade D, Russell NH, Walter RB, Estey EH, Westermann J, Gorner M, Benner A, Kramer A, Smith BD, Burnett AK, Thiede C, Rollig C, Ho AD, Ehninger G, Schlenk RF, Tallman MS, Levis MJ, Platzbecker U (2017) Characteristics and outcome of patients with therapy-related acute promyelocytic leukemia front-line treated with or without arsenic trioxide. Leukemia 31:2347–2354

    Article  CAS  Google Scholar 

  15. Lo-Coco F, Avvisati G, Vignetti M, Thiede C, Orlando SM, Iacobelli S, Ferrara F, Fazi P, Cicconi L, Di Bona E, Specchia G, Sica S, Divona M, Levis A, Fiedler W, Cerqui E, Breccia M, Fioritoni G, Salih HR, Cazzola M, Melillo L, Carella AM, Brandts CH, Morra E, von Lilienfeld-Toal M, Hertenstein B, Wattad M, Lubbert M, Hanel M, Schmitz N, Link H, Kropp MG, Rambaldi A, La Nasa G, Luppi M, Ciceri F, Finizio O, Venditti A, Fabbiano F, Dohner K, Sauer M, Ganser A, Amadori S, Mandelli F, Dohner H, Ehninger G, Schlenk RF, Platzbecker U, Gruppo Italiano Malattie Ematologiche dell’Adulto, German-Austrian Acute Myeloid Leukemia Study Group, Study Alliance Leukemia (2013) Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med 369:111–121

    Article  CAS  Google Scholar 

  16. Park DJ, Chumakov AM, Vuong PT, Chih DY, Gombart AF, Miller WH Jr, Koeffler HP (1999) CCAAT/enhancer binding protein epsilon is a potential retinoid target gene in acute promyelocytic leukemia treatment. J Clin Invest 103:1399–1408

    Article  CAS  Google Scholar 

  17. Mueller BU, Pabst T, Fos J, Petkovic V, Fey MF, Asou N, Buergi U, Tenen DG (2006) ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression. Blood 107:3330–3338

    Article  CAS  Google Scholar 

  18. Tomita A, Kiyoi H, Naoe T (2013) Mechanisms of action and resistance to all-trans retinoic acid (ATRA) and arsenic trioxide (As2O 3) in acute promyelocytic leukemia. Int J Hematol 97:717–725

    Article  CAS  Google Scholar 

  19. Nervi C, Ferrara FF, Fanelli M, Rippo MR, Tomassini B, Ferrucci PF, Ruthardt M, Gelmetti V, Gambacorti-Passerini C, Diverio D, Grignani F, Pelicci PG, Testi R (1998) Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARalpha fusion protein. Blood 92:2244–2251

    CAS  PubMed  Google Scholar 

  20. vom Baur E, Zechel C, Heery D, Heine MJ, Garnier JM, Vivat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R (1996) Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124

    Article  CAS  Google Scholar 

  21. Ablain J, Leiva M, Peres L, Fonsart J, Anthony E, de The H (2013) Uncoupling RARA transcriptional activation and degradation clarifies the bases for APL response to therapies. J Exp Med 210:647–653

    Article  CAS  Google Scholar 

  22. Mizushima N, Klionsky DJ (2007) Protein turnover via autophagy: implications for metabolism. Annu Rev Nutr 27:19–40

    Article  CAS  Google Scholar 

  23. Orfali N, McKenna SL, Cahill MR, Gudas LJ, Mongan NP (2014) Retinoid receptor signaling and autophagy in acute promyelocytic leukemia. Exp Cell Res 324:1–12

    Article  CAS  Google Scholar 

  24. Orfali N, O'Donovan TR, Nyhan MJ, Britschgi A, Tschan MP, Cahill MR, Mongan NP, Gudas LJ, McKenna SL (2015) Induction of autophagy is a key component of all-trans-retinoic acid-induced differentiation in leukemia cells and a potential target for pharmacologic modulation. Exp Hematol 43:781–93 e2

    Article  CAS  Google Scholar 

  25. Chen ZH, Wang WT, Huang W, Fang K, Sun YM, Liu SR, Luo XQ, Chen YQ (2017) The lncRNA HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway. Cell Death Differ 24:212–224

    Article  CAS  Google Scholar 

  26. Weidberg H, Shvets E, Shpilka T, Shimron F, Shinder V, Elazar Z (2010) LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. EMBO J 29:1792–1802

    Article  CAS  Google Scholar 

  27. Lihuan D, Jingcun Z, Ning J, Guozeng W, Yiwei C, Wei L, Jing Q, Yuanfang Z, Gang C (2014) Photodynamic therapy with the novel photosensitizer chlorophyllin f induces apoptosis and autophagy in human bladder cancer cells. Lasers Surg Med 46:319–334

    Article  Google Scholar 

  28. Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, Piqani B, Eisenhaure TM, Luo B, Grenier JK, Carpenter AE, Foo SY, Stewart SA, Stockwell BR, Hacohen N, Hahn WC, Lander ES, Sabatini DM, Root DE (2006) A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 124:1283–1298

    Article  CAS  Google Scholar 

  29. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162(1):156–159

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology, Weill Cornell Medicine, New York, NY, USA

    Nina Orfali, Dalyia Benjamin, Lorraine J. Gudas & Nigel P. Mongan

  2. Cork Cancer Research Centre, University College Cork, Cork, Ireland

    Nina Orfali, Tracey R. O’Donovan, Dalyia Benjamin, Mary Cahill & Sharon McKenna

  3. Department of Haematology, Cork University Hospital, Cork, Ireland

    Nina Orfali & Mary Cahill

  4. Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, Nottingham, UK

    Jennie N. Jeyapalan, Corinne L. Woodcock & Nigel P. Mongan

Authors
  1. Nina Orfali
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jennie N. Jeyapalan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Corinne L. Woodcock
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tracey R. O’Donovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dalyia Benjamin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mary Cahill
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sharon McKenna
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lorraine J. Gudas
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nigel P. Mongan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Nigel P. Mongan .

Editor information

Editors and Affiliations

  1. Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine, Columbia, SC, USA

    Swapan K. Ray

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Orfali, N. et al. (2019). Lentiviral-Mediated shRNA Approaches: Applications in Cellular Differentiation and Autophagy. In: Ray, S. (eds) Retinoid and Rexinoid Signaling . Methods in Molecular Biology, vol 2019. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9585-1_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9585-1_3

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9584-4

  • Online ISBN: 978-1-4939-9585-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation