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Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
2011 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 6, no 3, e17532- p.Article in journal (Refereed) Published
Abstract [en]

Replication-competent adenovirus type 5 (Ad5) vectors promise to be more efficient gene delivery vehicles than their replication-deficient counterparts, and chimeric Ad5 vectors that are capable of targeting CD46 are more effective than Ad5 vectors with native fibers. Although several strategies have been used to improve gene transduction and oncolysis, either by modifying their tropism or enhancing their replication capacity, some tumor cells are still relatively refractory to infection by chimeric Ad5. The oncolytic effects of the vectors are apparent in certain tumors but not in others. Here, we report the biological and oncolytic profiles of a replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells. CD46 was abundantly expressed in all cells studied; however, the transduction efficiency of RCAd11pGFP varied. RCAd11pGFP efficiently transduced HT-29, HCT-8, and LS174T cells, but it transduced T84 cells, derived from a colon cancer metastasis in the lung, less efficiently. Interestingly, RCAd11p replicated more rapidly in the T84 cells than in HCT-8 and LS174T cells and as rapidly as in HT-29 cells. Cell toxicity and proliferation assays indicated that RCAd11pGFP had the highest cell-killing activities in HT29 and T84 cells, the latter of which also expressed the highest levels of glycoproteins of the carcinoma embryonic antigen (CEA) family. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumors in xenograft mice treated with either RCAd11pGFP or Ad11pwt compared to untreated controls. Thus, RCAd11pGFP has a potent cytotoxic effect on colon carcinoma cells.

Place, publisher, year, edition, pages
2011. Vol. 6, no 3, e17532- p.
Keyword [en]
gene-transfer vector; carcinoembryonic antigen; nucleotide-sequence; colorectal-cancer; cellular receptor; cd46 receptor; therapy; virus; expression; serotype-3
National Category
Biomedical Laboratory Science/Technology
URN: urn:nbn:se:umu:diva-41973DOI: 10.1371/journal.pone.0017532PubMedID: 21455297OAI: diva2:408213
Available from: 2011-04-04 Created: 2011-04-04 Last updated: 2011-12-22Bibliographically approved
In thesis
1. Replication-competent adenovirus 11p vector as a new oncolytic agent
Open this publication in new window or tab >>Replication-competent adenovirus 11p vector as a new oncolytic agent
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Human adenoviruses (Ads) as vectors have been studied for cancer gene therapy for several decades due to their ability to shut down host cell replication and lyse tumour cells. Ad5 of species C is commonly used as a replication-defective or a replication-competent vector. However, many tumour cells are relatively refractory to infection by Ad5 since the cells lack the viral receptor CAR. Thus, species B Ads are becoming more important as alternative vectors since they use CD46 as primary receptor, the expression of which is up-regulated on many tumour cell surfaces, and they also have low seroprevalence in humans. Although Ad3, Ad7, Ad11 and Ad35 have been altered to become replication-defective vectors, investigations based on replicating adenovirus vectors are still warranted.  

The major aim of this thesis has been to characterize the transduction efficacy and oncolytic effect of the replication-competent adenovirus 11pGFP vector (RCAd11pGFP) in human solid tumour cell lines. Evaluation of the vector would ultimately help us to understand whether the tumour cells affect virus replication and whether the vector replicates differently in tumour cells and in untransformed diploid cells, and would eventually lead to development of more potent oncolytic adenoviruses for treatment of human cancers.

The Ad11-based vector RCAd11pGFP consists of the entire Ad11p genome with a green fluorescence protein (GFP) expression cassette inserted. RCAd11pGFP shows all the characteristics of the wild-type virus and expresses GFP in cells four hours p.i. Antisera raised against Ad11p virions and hexons were able to neutralize RCAd11pGFP infection but antiserum raised against the Ad11p fibre knob could not. The infection is reduced by 90% but the fibre knob antiserum cannot completely block virus infection. Initial screening of the infection capacity of five wild-type adenoviruses in four colon cancer cell lines revealed that Ad11p, Ad11a and Ad35 of species B, showed similar replication kinetics but Ad5 showed delayed onset of virus replication in comparison to species B Ads. These data support the use of Ad11p as an alternative vector for treatment of colon cancer.

The transduction efficiency of RCAd11pGFP in colon cancer and prostate cancer cell lines was studied using flow cytometry assay (FACS), and this showed that the cytolytic effect was not always in accordance with GFP expression. Toxicity assay and virus one-step replication assay showed that RCAd11pGFP replicates in highly tumorigenic cell lines (HT29, T84 and PC-3) to a greater extent than less tumorigenic cell lines (LS174T, HCT-8, DU145 and LNCaP cells), even though the latter showed relatively high GFP expression. This initial finding led to the subsequent discovery of CEACAM-family molecules, which were highly expressed in HT29 and T84 cells. Interestingly, the Ad5 wild-type virus did not manifest the same tumour-specific replication that RCAd11pGFP did in the cell lines studied.

Furthermore, we investigated the influence of tumour markers for RCAd11pGFP replication in colon cancer cells. A double-staining FACS assay for detecting members of CEACAM-family molecules was established and we found that the levels of CEACAM6 were up-regulated in the cells infected by RCAd11pGFP or Ad11pwt relative to uninfected cells. However, this virus replication could not be suppressed by CEACEA6 siRNA. Our results indicate that several tumour markers or factors might be involved in promoting propagation of the virus.

In vivo experiments showed significant growth inhibition of T84 and HT-29 tumours in xenograft mice treated with either RCAd11pGFP or Ad11pwt, compared to untreated controls. Furthermore, the role of the anti-tumour effect of RCAd11pGFP was also confirmed in PC3 prostate tumours in BALB/c mice.

In conclusion, the novel RCAd11pGFP vector was shown to have an anti-tumour effect in vitro and in vivo. This tumour-killing effect could be enhanced in highly tumorogenic cells through virus replication. Consequently, RCAd11p may lead to development of a more potent and useful vector for human cancer therapy.  

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2011. 74 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1473
Virologi, Adenovirus, Vector
National Category
Medical and Health Sciences
Research subject
urn:nbn:se:umu:diva-50773 (URN)978-91-7459-333-4 (ISBN)
Public defence
2012-01-20, Astrid Fagreussalen, by 6, E04 Unod, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Available from: 2011-12-22 Created: 2011-12-21 Last updated: 2011-12-22Bibliographically approved

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