Skip to main content

Advertisement

SpringerLink
Log in

Novel lentiviral-inducible transgene expression systems and versatile single-plasmid reporters for in vitro and in vivo cancer biology studies

  • Original Article
  • Published:
Cancer Gene Therapy Submit manuscript

Abstract

Many of the cancer cell lines derived from solid tumors are difficult to transfect using commonly established transfection approaches. This hurdle for some DNA transfection systems has hindered cancer biology studies. Moreover, there are limited tools for studying pathway activities. Therefore, highly efficient improved gene transfer and versatile genetic tools are required. In this study, we established and developed a comprehensive set of new lentiviral tools to study gene functions and pathway activities. Using the optimized conditions, cancer cell lines achieved >90% transduction efficiency. Novel lentiviral doxycycline-regulated pTet-IRES-EGFP (pTIE) systems for transgene expression and TRE reporters used for pathway activity determination were developed and tested. The pTIE Tet-Off system showed in vitro doxycycline-sensitive responses with low or undetectable leakage of protein expression and in vivo tumor suppression as illustrated using candidate tumor suppressors, Fibulin-2 and THY1. In contrast, the Tet-On system showed dose-dependent responses. The pTRE-EGFP (pTE) and pTRE-FLuc-EF1α-RLuc (pT-FER) reporters with the NFκB p65 subunit consensus sequence showed GFP and firefly luciferase responses, which were directly correlated with TNFα stimulation, respectively. Taken together, these newly developed lentiviral systems provide versatile in vitro and in vivo platforms to strengthen our capabilities for cancer biology studies.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Geiling B, Vandal G, Posner AR, de Bruyns A, Dutchak KL, Garnett S et al. A modular lentiviral and retroviral construction system to rapidly generate vectors for gene expression and gene knockdown in vitro and in vivo. PLoS One 2013; 8: e76279.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Cepko CL, Roberts BE, Mulligan RC . Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 1984; 37: 1053–1062.

    Article  CAS  PubMed  Google Scholar 

  3. Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 1996; 272: 263–267.

    Article  CAS  PubMed  Google Scholar 

  4. Logan AC, Lutzko C, Kohn DB . Advances in lentiviral vector design for gene-modification of hematopoietic stem cells. Curr Opin Biotechnol 2002; 13: 429–436.

    Article  CAS  PubMed  Google Scholar 

  5. Park F, Ohashi K, Chiu W, Naldini L, Kay M . Efficient lentiviral transduction of liver requires cell cycling in vivo. Nat Genet 2000; 24: 49–52.

    Article  CAS  PubMed  Google Scholar 

  6. Olsen JC . Gene transfer vectors derived from equine infectious anemia virus. Gene Ther 1998; 5: 1481–1487.

    Article  CAS  PubMed  Google Scholar 

  7. Poeschla EM, Wong-Staal F, Looney DJ . Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors. Nat Med 1998; 4: 354–357.

    Article  CAS  PubMed  Google Scholar 

  8. Miyoshi H, Blömer U, Takahashi M, Gage FH, Verma IM . Development of a self-inactivating lentivirus vector. J Virol 1998; 72: 8150–8157.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72: 9873–9880.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D et al. A third-generation lentivirus vector with a conditional packaging system. J Virol 1998; 72: 8463–8471.

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Federico M . Lentiviruses as gene delivery vectors. Curr Opin Biotechnol 1999; 10: 448–453.

    Article  CAS  PubMed  Google Scholar 

  12. Law EWL, Cheung aKL, Kashuba VI, Pavlova TV, Zabarovsky ER, Lung HL et al. Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma. Oncogene 2012; 31: 728–738.

    Article  CAS  PubMed  Google Scholar 

  13. Wong AMG, Kong KL, Chen L, Liu M, Wong AMG, Zhu C et al. Characterization of CACNA2D3 as a putative tumor suppressor gene in the development and progression of nasopharyngeal carcinoma. Int J Cancer 2013; 133: 2284–2295.

    Article  CAS  PubMed  Google Scholar 

  14. Protopopov AI, Li J, Winberg G, Gizatullin RZ, Kashuba VI, Klein G et al. Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21. J Gene Med 2002; 4: 397–406.

    Article  CAS  PubMed  Google Scholar 

  15. Chan KC, Ko JMY, Lung HL, Sedlacek R, Zhang Z-F, Luo D-Z et al. Catalytic activity of Matrix metalloproteinase-19 is essential for tumor suppressor and anti-angiogenic activities in nasopharyngeal carcinoma. Int J Cancer 2011; 129: 1826–1837.

    Article  CAS  PubMed  Google Scholar 

  16. Cheung AKL, Ko JMY, Lung HL, Chan KW, Stanbridge EJ, Zabarovsky E et al. Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-kappaB-mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis. Proc Natl Acad Sci USA 2011; 108: 8390–8395.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lung HL, Lo PHY, Xie D, Apte SS, Cheung AKL, Cheng Y et al. Characterization of a novel epigenetically-silenced, growth-suppressive gene, ADAMTS9, and its association with lymph node metastases in nasopharyngeal carcinoma. Int J Cancer 2008; 123: 401–408.

    Article  CAS  PubMed  Google Scholar 

  18. Huang Z, Cheng Y, Chiu PM, Cheung FMF, Nicholls JM, Kwong DL-W et al. Tumor suppressor Alpha B-crystallin (CRYAB) associates with the cadherin/catenin adherens junction and impairs NPC progression-associated properties. Oncogene 2012; 31: 3709–3720.

    Article  CAS  PubMed  Google Scholar 

  19. Cheung AKL, Lung HL, Hung SC, Law EWL, Cheng Y, Yau WL et al. Functional analysis of a cell cycle-associated, tumor-suppressive gene, protein tyrosine phosphatase receptor type G, in nasopharyngeal carcinoma. Cancer Res 2008; 68: 8137–8145.

    Article  CAS  PubMed  Google Scholar 

  20. Lung HL, Cheung AKL, Cheng Y, Kwong FM, Lo PHY, Law EWL et al. Functional characterization of THY1 as a tumor suppressor gene with antiinvasive activity in nasopharyngeal carcinoma. Int J Cancer 2010; 127: 304–312.

    CAS  PubMed  Google Scholar 

  21. Lung HL, Cheung AKL, Xie D, Cheng Y, Kwong FM, Murakami Y et al. TSLC1 is a tumor suppressor gene associated with metastasis in nasopharyngeal carcinoma. Cancer Res 2006; 66: 9385–9392.

    Article  CAS  PubMed  Google Scholar 

  22. Cheung AKL, Lung HL, Ko JMY, Cheng Y, Stanbridge EJ, Zabarovsky ER et al. Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma. Proc Natl Acad Sci USA 2009; 106: 14478–14483.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Glaser R, Zhang HY, Yao KT, Zhu HC, Wang FX, Li GY et al. Two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus that were derived from nasopharyngeal carcinomas. Proc Natl Acad Sci USA 1989; 86: 9524–9528.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Yao KT, Zhang HY, Zhu HC, Wang FX, Li GY, Wen DS et al. Establishment and characterization of two epithelial tumor cell lines (HNE-1 and HONE-1) latently infected with Epstein-Barr virus and derived from nasopharyngeal carcinomas. Int J Cancer 1990; 45: 83–89.

    Article  CAS  PubMed  Google Scholar 

  25. Cancer Institute Laboratory of Cell Biology Chinese Academy of Medical Science Beijing and Chung Shan Medical College. Establishment of an epitheloid cell line and a fusiform cell line from a patient with nasopharyngeal carcinoma. Sci Sin 1978; 21: 127–134.

    Google Scholar 

  26. Sizhong Z, Xiukung G, Yi Z . Cytogenetic studies on an epithelial cell line derived from poorly differentiated nasopharyngeal carcinoma. Int J Cancer 1983; 31: 587–590.

    Article  CAS  PubMed  Google Scholar 

  27. Rasheed S, Nelson-Rees WA, Toth EM, Arnstein P, Gardner MB . Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 1974; 33: 1027–1033.

    Article  CAS  PubMed  Google Scholar 

  28. Gluzman Y . SV40-transformed simian cells support the replication of early SV40 mutants. Cell 1981; 23: 175–182.

    Article  CAS  PubMed  Google Scholar 

  29. Graham FL, van der Eb AJ . A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973; 52: 456–467.

    Article  CAS  PubMed  Google Scholar 

  30. Scherer WF, Syverton JT, Gey GO . Studies on the propagation in vitro of poliomyelitis viruses. IV. Viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 1953; 97: 695–710.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lieber M, Smith B, Szakal A, Nelson-Rees W, Todaro G . A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 1976; 17: 62–70.

    Article  CAS  PubMed  Google Scholar 

  32. Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS et al. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer 1999; 83: 121–126.

    Article  CAS  PubMed  Google Scholar 

  33. Hu CP, Hsieh HG, Chien KY, Wang PY, Wang CI, Chen CY et al. Biologic properties of three newly established human esophageal carcinoma cell lines. J Natl Cancer Inst 1984; 72: 577–583.

    CAS  PubMed  Google Scholar 

  34. Mok CH, Chew EC, Riches DJ, Lee JC, Huang DP, Hadgis C et al. Biological characteristics of a newly established human oesophageal carcinoma cell line. Anticancer Res 7: 409–415.

  35. Lung HL, Bangarusamy DK, Xie D, Cheung AKL, Cheng Y, Kumaran MK et al. THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma. Oncogene 2005; 24: 6525–6532.

    Article  CAS  PubMed  Google Scholar 

  36. Lee M-H, Padmashali R, Andreadis ST . JNK1 is required for lentivirus entry and gene transfer. J Virol 2011; 85: 2657–2665.

    Article  CAS  PubMed  Google Scholar 

  37. Lee M-H, Padmashali R, Koria P, Andreadis ST . JNK regulates binding of alpha-catenin to adherens junctions and cell-cell adhesion. FASEB J 2011; 25: 613–623.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Padmashali R, You H, Karnik N, Lei P, Andreadis ST . Adherens junction formation inhibits lentivirus entry and gene transfer. PLoS One 2013; 8: e79265.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Yoon M, Spear PG . Disruption of adherens junctions liberates nectin-1 to serve as receptor for herpes simplex virus and pseudorabies virus entry. J Virol 2002; 76: 7203–7208.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Mao G, Marotta F, Yu J, Zhou L, Yu Y, Wang L et al. DNA context and promoter activity affect gene expression in lentiviral vectors. Acta Biomed 2008; 79: 192–196.

    CAS  PubMed  Google Scholar 

  41. Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren B-Z et al. Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 2010; 5: e10611.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Lung HL, Man OY, Yeung MC, Ko JMY, Cheung AKL, Law EWL et al. SAA1 polymorphisms are associated with variation in antiangiogenic and tumor-suppressive activities in nasopharyngeal carcinoma. Oncogene 2015; 34: 878–889.

    Article  CAS  PubMed  Google Scholar 

  43. Lo PHY, Lung HL, Cheung AKL, Apte SS, Chan KW, Kwong FM et al. Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis. Cancer Res 2010; 70: 5567–5576.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li J, Wang F, Protopopov A, Malyukova A, Kashuba V, Minna JD et al. Inactivation of RASSF1C during in vivo tumor growth identifies it as a tumor suppressor gene. Oncogene 2004; 23: 5941–5949.

    Article  CAS  PubMed  Google Scholar 

  45. Rubins JB, Charboneau D, Alter MD, Bitterman PB, Kratzke RA . Inhibition of mesothelioma cell growth in vitro by doxycycline. J Lab Clin Med 2001; 138: 101–106.

    Article  CAS  PubMed  Google Scholar 

  46. Haack K, Cockrell AS, Ma H, Israeli D, Ho SN, McCown TJ et al. Transactivator and structurally optimized inducible lentiviral vectors. Mol Ther 2004; 10: 585–596.

    Article  CAS  PubMed  Google Scholar 

  47. Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell 2006; 124: 1283–1298.

    Article  CAS  PubMed  Google Scholar 

  48. Wiederschain D, Wee S, Chen L, Loo A, Yang G, Huang A et al. Single-vector inducible lentiviral RNAi system for oncology target validation. Cell Cycle 2009; 8: 498–504.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Faculty Core Facility of the University of Hong Kong for the technical support and equipment access. We thank Wai Yin Chau for technical assistance of mouse experiment. We also thank Professor Didier Trono for providing pWPI, psPAX2 and pMD2G plasmids, and Professor Eugene Zabarovsky for providing pETE-Bsd and pLNCtTA-hCMV plasmids. This work was funded by the Research Grants Council Area of Excellence scheme in Hong Kong (AoE/M-06/08) to MLL.

Author information

Author notes

  1. W H Shuen

    Present address: 4Current address: Division of Medical Oncology, National Cancer Centre Singapore, 11 Hospital Drive, Singapore 169610, Singapore,

Authors and Affiliations

  1. Department of Clinical Oncology, University of Hong Kong, Hong Kong (SAR), China,

    W H Shuen, R Kan, Z Yu, H L Lung & M L Lung

  2. Center for Cancer Research, University of Hong Kong, Hong Kong (SAR), China,

    H L Lung & M L Lung

  3. Center for Nasopharyngeal Carcinoma Research, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong (SAR), China

    H L Lung & M L Lung

Authors
  1. W H Shuen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. R Kan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Z Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. H L Lung
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M L Lung
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M L Lung.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Cancer Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shuen, W., Kan, R., Yu, Z. et al. Novel lentiviral-inducible transgene expression systems and versatile single-plasmid reporters for in vitro and in vivo cancer biology studies. Cancer Gene Ther 22, 207–214 (2015). https://doi.org/10.1038/cgt.2015.9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/cgt.2015.9

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation