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Infectivity enhanced, hTERT promoter-based conditionally replicative adenoviruses are useful for SCLC treatment

Cancer Gene Therapy volume 12, pages 737–748 (2005)Cite this article

Abstract

Treatment of advanced small-cell lung cancer (SCLC) remains one of the major challenges in current medicine because of the high morbidity and mortality of the disease. Advanced stage lung cancer is refractory to conventional therapies and it also has an extremely poor prognosis. As a result, new therapeutic approaches are needed. Telomere maintenance to the regulation of replicative lifespan strongly implies that alterations in telomere biology play an important role during malignant transformation. Cancers that exhibit high levels of telomerase activity, such as all of the SCLC, were examined in a previous study. In this study, we turned the expression of human telomerase reverse transcriptase (hTERT) by tumors to a therapeutic advantage using a conditionally replication-competent adenovirus (CRAd) in which the expression of E1 (early region 1) is controlled by the hTERT promoter. This virus achieved good levels of viral replication in SCLC cells and induced a substantial anticancer effect in vitro and in vivo. As a further enhancement, the cancer cell killing effect was improved with a tropism modification of the virus to express the knob domain of Ad3 (serotype 3 adenovirus), and this improved infectivity for cancer cells. Conversely, the hTERT promoter has low activity in normal tissues, and the CRAd caused no damage to normal lung fibroblast cells. Since the telomerase activity is common in many types of cancers, these CRAds may be applicable to a wide range of tumors. We concluded that the use of hTERT promoter-based CRAds may be a potentially effective strategy for cancer treatment.

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Abbreviations

SCLC:

small-cell lung cancer

hTERT:

human telomerase reverse transcriptase

TRF:

telomere restriction fragment

TRAP:

telomeric repeat amplification protocol

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

TERC:

telomerase RNA component

GRP:

gastrin-releasing peptide

CAR:

coxsackie and adenovirus receptor

Ad5:

serotype 5 adenovirus

Ad3:

serotype 3 adenovirus

Ad5/3:

Ad5 containing a chimeric fiber protein possessing the Ad3 knob

CRAd:

conditionally replicative adenovirus

E1:

early region 1

ITR:

inverted terminal repeat

RT-PCR:

reverse transcription-PCR

CMV:

cytomegalovirus

MOI:

multiplicity of infection

TCID50:

tissue culture infectious dose 50

VP:

viral particle

PFU:

plaque forming unit

References

  1. Blackburn EH . Switching and signaling at the telomere. Cell. 2001;106:661–673.

    Article  CAS  PubMed  Google Scholar 

  2. McEachern MJ, Krauskopf A, Blackburn EH . Telomeres and their control. Annu Rev Genet. 2000;34:331–358.

    Article  CAS  PubMed  Google Scholar 

  3. Meyne J, Ratliff RL, Moyzis RK . Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Natl Acad Sci USA. 1989;86:7049–7053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Moyzis RK, Buckingham JM, Cram LS, et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci USA. 1988;85:6622–6626.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. de Lange T . Protection of mammalian telomeres. Oncogene. 2002;21:532–540.

    Article  CAS  PubMed  Google Scholar 

  6. Chong L, van Steensel B, Broccoli D, et al. A human telomeric protein. Science. 1995;270:1663–1667.

    Article  CAS  PubMed  Google Scholar 

  7. Broccoli D, Smogorzewska A, Chong L, de Lange T . Human telomeres contain two distinct Myb-related proteins, TRF1 and TRF2. Nat Genet. 1997;17:231–235.

    Article  CAS  PubMed  Google Scholar 

  8. Bilaud T, Brun C, Ancelin K, et al. Telomeric localization of TRF2, a novel human telobox protein. Nat Genet. 1997;17:236–239.

    Article  CAS  PubMed  Google Scholar 

  9. Feng J, Funk WD, Wang SS, et al. The RNA component of human telomerase. Science. 1995;269:1236–1241.

    Article  CAS  PubMed  Google Scholar 

  10. Lingner J, Hughes TR, Shevchenko A, et al. Reverse transcriptase motifs in the catalytic subunit of telomerase. Science. 1997;276:561–567.

    Article  CAS  PubMed  Google Scholar 

  11. Nugent CI, Lundblad V . The telomerase reverse transcriptase: components and regulation. Genes Dev. 1998;12:1073–1085.

    Article  CAS  PubMed  Google Scholar 

  12. Kim NW, Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994;266:2011–2015.

    Article  CAS  PubMed  Google Scholar 

  13. Shay JW, Bacchetti S . A survey of telomerase activity in human cancer. Eur J Cancer. 1997;33:787–791.

    Article  CAS  PubMed  Google Scholar 

  14. Bryan TM, Marusic L, Bacchetti S, Namba M, Reddel RR . The telomere lengthening mechanism in telomerase-negative immortal human cells does not involve the telomerase RNA subunit. Hum Mol Genet. 1997;6:921–926.

    Article  CAS  PubMed  Google Scholar 

  15. Kim NW . Clinical implications of telomerase in cancer. Eur J Cancer. 1997;33:781–786.

    Article  CAS  PubMed  Google Scholar 

  16. Hiyama K, Hiyama E, Ishioka S, et al. Telomerase activity in small-cell and non-small-cell lung cancers. J Natl Cancer Inst. 1995;87:895–902.

    Article  CAS  PubMed  Google Scholar 

  17. Ganly I, Kirn D, Eckhardt G, et al. A phase I study of Onyx-015, an E1B attenuated adenovirus, administered intratumorally to patients with recurrent head and neck cancer. Clin Cancer Res. 2000;6:798–806.

    CAS  PubMed  Google Scholar 

  18. DeWeese TL, van der Poel H, Li S, et al. A phase I trial of CV706, a replication-competent, PSA selective oncolytic adenovirus, for the treatment of locally recurrent prostate cancer following radiation therapy. Cancer Res. 2001;61:7464–7472.

    CAS  PubMed  Google Scholar 

  19. Benjamin R, Helman L, Meyers P, Reaman G . A phase I/II dose escalation and activity study of intravenous injections of OCaP1 for subjects with refractory osteosarcoma metastatic to lung. Hum Gene Ther. 2001;12:1591–1593.

    CAS  PubMed  Google Scholar 

  20. Zabner J, Freimuth P, Puga A, Fabrega A, Welsh MJ . Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J Clin Invest. 1997;100:1144–1149.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kaner RJ, Worgall S, Leopold PL, et al. Modification of the genetic program of human alveolar macrophages by adenovirus vectors in vitro is feasible but inefficient, limited in part by the low level of expression of the coxsackie/adenovirus receptor. Am J Respir Cell Mol Biol. 1999;20:361–370.

    Article  CAS  PubMed  Google Scholar 

  22. Nalbantoglu J, Larochelle N, Wolf E, et al. Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle. J Virol. 2001;75:4276–4282.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Takayama K, Reynolds PN, Short JJ, et al. A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism. Virology. 2003;309:282–293.

    Article  CAS  PubMed  Google Scholar 

  24. Miller CR, Buchsbaum DJ, Reynolds PN, et al. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer. Cancer Res. 1998;58:5738–5748.

    CAS  PubMed  Google Scholar 

  25. Hemmi S, Geertsen R, Mezzacasa A, Peter I, Dummer R . The presence of human coxsackievirus and adenovirus receptor is associated with efficient adenovirus-mediated transgene expression in human melanoma cell cultures. Hum Gene Ther. 1998;9:2363–2373.

    Article  CAS  PubMed  Google Scholar 

  26. Kanerva A, Mikheeva GV, Krasnykh V, et al. Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res. 2002;8:275–280.

    CAS  PubMed  Google Scholar 

  27. He TC, Zhou S, da Costa LT, et al. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998;95:2509–2514.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dmitriev I, Krasnykh V, Miller CR, et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol. 1998;72:9706–9713.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Takakura M, Kyo S, Kanaya T, et al. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells. Cancer Res. 1999;59:551–557.

    CAS  PubMed  Google Scholar 

  30. Yamamoto M, Alemany R, Adachi Y, Grizzle WE, Curiel DT . Characterization of the cyclooxygenase-2 promoter in an adenoviral vector and its application for the mitigation of toxicity in suicide gene therapy of gastrointestinal cancers. Mol Ther. 2001;3:385–394.

    Article  CAS  PubMed  Google Scholar 

  31. Krasnykh VN, Mikheeva GV, Douglas JT, Curiel DT . Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism. J Virol. 1996;70:6839–6846.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Bett AJ, Haddara W, Prevec L, Graham FL . An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. Proc Natl Acad Sci USA. 1994;91:8802–8806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Adachi Y, Reynolds PN, Yamamoto M, et al. A midkine promoter-based conditionally replicative adenovirus for treatment of pediatric solid tumors and bone marrow tumor purging. Cancer Res. 2001;61:7882–7888.

    CAS  PubMed  Google Scholar 

  34. Maizel Jr JV, White DO, Scharff MD . The polypeptides of adenovirus. I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12. Virology. 1968;36:115–125.

    Article  CAS  PubMed  Google Scholar 

  35. Nyberg-Hoffman C, Shabram P, Li W, Giroux D, Aguilar-Cordova E . Sensitivity and reproducibility in adenoviral infectious titer determination. Nat Med. 1997;3:808–811.

    Article  CAS  PubMed  Google Scholar 

  36. Dhaene K, Wauters J, Weyn B, Timmermans JP, van Marck E . Expression profile of telomerase subunits in human pleural mesothelioma. J Pathol. 2000;190:80–85.

    Article  CAS  PubMed  Google Scholar 

  37. Tsutsui J, Kadomatsu K, Matsubara S, et al. A new family of heparin-binding growth/differentiation factors: increased midkine expression in Wilms' tumor and other human carcinomas. Cancer Res. 1993;53:1281–1285.

    CAS  PubMed  Google Scholar 

  38. Spindel ER, Chin WW, Price J, et al. Cloning and characterization of cDNAs encoding human gastrin-releasing peptide. Proc Natl Acad Sci USA. 1984;81:5699–5703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sausville EA, Lebacq-Verheyden AM, Spindel ER, et al. Expression of the gastrin-releasing peptide gene in human small cell lung cancer. Evidence for alternative processing resulting in three distinct mRNAs. J Biol Chem. 1986;261:2451–2457.

    CAS  PubMed  Google Scholar 

  40. Niiyama H, Mizumoto K, Sato N, et al. Quantitative analysis of hTERT mRNA expression in colorectal cancer. Am J Gastroenterol. 2001;96:1895–1900.

    Article  CAS  PubMed  Google Scholar 

  41. Haviv YS, Blackwell JL, Kanerva A, et al. Adenoviral gene therapy for renal cancer requires retargeting to alternative cellular receptors. Cancer Res. 2002;62:4273–4281.

    CAS  PubMed  Google Scholar 

  42. Takayama K, Ueno H, Nakanishi Y, et al. Suppression of tumor angiogenesis and growth by gene transfer of a soluble form of vascular endothelial growth factor receptor into a remote organ. Cancer Res. 2000;60:2169–2177.

    CAS  PubMed  Google Scholar 

  43. Wege H, Chui MS, Le HT, Tran JM, Zern MA . SYBR Green real-time telomeric repeat amplification protocol for the rapid quantification of telomerase activity. Nucleic Acids Res. 2003;31:E3–3.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Matsubara S, Wada Y, Gardner TA, et al. A conditional replication-competent adenoviral vector, Ad-OC-E1a, to cotarget prostate cancer and bone stroma in an experimental model of androgen-independent prostate cancer bone metastasis. Cancer Res. 2001;61:6012–6019.

    CAS  PubMed  Google Scholar 

  45. Nettelbeck DM, Rivera AA, Balague C, Alemany R, Curiel DT . Novel oncolytic adenoviruses targeted to melanoma: specific viral replication and cytolysis by expression of E1A mutants from the tyrosinase enhancer/promoter. Cancer Res. 2002;62:4663–4670.

    CAS  PubMed  Google Scholar 

  46. Kurihara T, Brough DE, Kovesdi I, Kufe DW . Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen. J Clin Invest. 2000;106:763–771.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Iwao T, Hiyama E, Yokoyama T, et al. Telomerase activity for the preoperative diagnosis of pancreatic cancer. J Natl Cancer Inst. 1997;89:1621–1623.

    Article  CAS  PubMed  Google Scholar 

  48. Hiyama K, Ishioka S, Shay JW, et al. Telomerase activity as a novel marker of lung cancer and immune-associated lung diseases. Int J Mol Med. 1998;1:545–549.

    CAS  PubMed  Google Scholar 

  49. Sen S, Reddy VG, Khanna N, et al. A comparative study of telomerase activity in sputum, bronchial washing and biopsy specimens of lung cancer. Lung Cancer. 2001;33:41–49.

    Article  CAS  PubMed  Google Scholar 

  50. Yahata N, Ohyashiki K, Ohyashiki JH, et al. Telomerase activity in lung cancer cells obtained from bronchial washings. J Natl Cancer Inst. 1998;90:684–690.

    Article  CAS  PubMed  Google Scholar 

  51. Yang CT, Lee MH, Lan RS, Chen JK . Telomerase activity in pleural effusions: diagnostic significance. J Clin Oncol. 1998;16:567–573.

    Article  CAS  PubMed  Google Scholar 

  52. Takeda T, Inaba H, Yamazaki M, et al. Tumor-specific gene therapy for undifferentiated thyroid carcinoma utilizing the telomerase reverse transcriptase promoter. J Clin Endocrinol Metab. 2003;88:3531–3538.

    Article  CAS  PubMed  Google Scholar 

  53. Horikawa I, Cable PL, Afshari C, Barrett JC . Cloning and characterization of the promoter region of human telomerase reverse transcriptase gene. Cancer Res. 1999;59:826–830.

    CAS  PubMed  Google Scholar 

  54. Cong YS, Wen J, Bacchetti S . The human telomerase catalytic subunit hTERT: organization of the gene and characterization of the promoter. Hum Mol Genet. 1999;8:137–142.

    Article  CAS  PubMed  Google Scholar 

  55. Wu KJ, Grandori C, Amacker M, et al. Direct activation of TERT transcription by c-MYC. Nat Genet. 1999;21:220–224.

    Article  CAS  PubMed  Google Scholar 

  56. Oh S, Song YH, Kim UJ, Yim J, Kim TK . In vivo and in vitro analyses of Myc for differential promoter activities of the human telomerase (hTERT) gene in normal and tumor cells. Biochem Biophys Res Commun. 1999;263:361–365.

    Article  CAS  PubMed  Google Scholar 

  57. Kyo S, Takakura M, Taira T, et al. Sp1 cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene (hTERT). Nucleic Acids Res. 2000;28:669–677.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kyo S, Takakura M, Kanaya T, et al. Estrogen activates telomerase. Cancer Res. 1999;59:5917–5921.

    CAS  PubMed  Google Scholar 

  59. Misiti S, Nanni S, Fontemaggi G, et al. Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells. Mol Cell Biol. 2000;20:3764–3771.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kanaya T, Kyo S, Hamada K, et al. Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. Clin Cancer Res. 2000;6:1239–1247.

    CAS  PubMed  Google Scholar 

  61. Oh S, Song Y, Yim J, Kim TK . The Wilms' tumor 1 tumor suppressor gene represses transcription of the human telo1merase reverse transcriptase gene. J Biol Chem. 1999;274:37473–37478.

    Article  CAS  PubMed  Google Scholar 

  62. Gunes C, Lichtsteiner S, Vasserot AP, Englert C . Expression of the hTERT gene is regulated at the level of transcriptional initiation and repressed by Mad1. Cancer Res. 2000;60:2116–2121.

    CAS  PubMed  Google Scholar 

  63. Wirth T, Zender L, Schulte B, et al. A telomerase-dependent conditionally replicating adenovirus for selective treatment of cancer. Cancer Res. 2003;63:3181–3188.

    CAS  PubMed  Google Scholar 

  64. Huang TG, Savontaus MJ, Shinozaki K, Sauter BV, Woo SL . Telomerase-dependent oncolytic adenovirus for cancer treatment. Gene Therapy. 2003;10:1241–1247.

    Article  CAS  PubMed  Google Scholar 

  65. Cripe TP, Dunphy EJ, Holub AD, et al. Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells. Cancer Res. 2001;61:2953–2960.

    CAS  PubMed  Google Scholar 

  66. Davidoff AM, Stevenson SC, McClelland A, Shochat SJ, Vanin EF . Enhanced neuroblastoma transduction for an improved antitumor vaccine. J Surg Res. 1999;83:95–99.

    Article  CAS  PubMed  Google Scholar 

  67. Fechner H, Wang X, Wang H, et al. Trans-complementation of vector replication versus Coxsackie-adenovirus-receptor overexpression to improve transgene expression in poorly permissive cancer cells. Gene Therapy. 2000;7:1954–1968.

    Article  CAS  PubMed  Google Scholar 

  68. Kelly FJ, Miller CR, Buchsbaum DJ, et al. Selectivity of TAG-72-targeted adenovirus gene transfer to primary ovarian carcinoma cells versus autologous mesothelial cells in vitro. Clin Cancer Res. 2000;6:4323–4333.

    CAS  PubMed  Google Scholar 

  69. Li Y, Pong RC, Bergelson JM, et al. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res. 1999;59:325–330.

    CAS  PubMed  Google Scholar 

  70. Okegawa T, Li Y, Pong RC, et al. The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy. Cancer Res. 2000;60:5031–5036.

    CAS  PubMed  Google Scholar 

  71. Kawakami Y, Li H, Lam JT, et al. Substitution of the adenovirus serotype 5 knob with a serotype 3 knob enhances multiple steps in virus replication. Cancer Res. 2003;63:1262–1269.

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr K Mizumoto and Mrs S Nishio (Kyushu University, Fukuoka, Japan) for their excellent technical support and expert advice in performing the TRAP assay. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Health, Labour and Welfare of Japan (to KT), and Grant of Clinical Research Foundation (to KT).

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Authors and Affiliations

  1. Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Maidashi, Fukuoka, Japan

    Junji Uchino, Koichi Takayama, Akiko Harada, Hiroyuki Inoue & Yoichi Nakanishi

  2. Division of Human Gene Therapy, Departments of Medicine, Pathology and Surgery, Gene Therapy Center, The University of Alabama at Birmingham, Birmingham, Alabama, USA

    Koichi Takayama, Yosuke Kawakami & David T Curiel

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  1. Junji Uchino
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Correspondence to Koichi Takayama.

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Uchino, J., Takayama, K., Harada, A. et al. Infectivity enhanced, hTERT promoter-based conditionally replicative adenoviruses are useful for SCLC treatment. Cancer Gene Ther 12, 737–748 (2005). https://doi.org/10.1038/sj.cgt.7700838

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