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Replication-competent adenovirus 11p vector as a new oncolytic agent
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology. (Ya-Fang Mei)
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.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1473
Keyword [en]
Virologi, Adenovirus, Vector
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-50773ISBN: 978-91-7459-333-4 (print)OAI: oai:DiVA.org:umu-50773DiVA: diva2:468690
Public defence
2012-01-20, Astrid Fagreussalen, by 6, E04 Unod, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-12-22 Created: 2011-12-21 Last updated: 2011-12-22Bibliographically approved
List of papers
1. Completely replication-competent adenovirus 11p GFP (RCAd11pGFP) vector with an insertion upstream of the full-length E1 region
Open this publication in new window or tab >>Completely replication-competent adenovirus 11p GFP (RCAd11pGFP) vector with an insertion upstream of the full-length E1 region
(English)Article in journal (Other academic) Submitted
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-50799 (URN)
Available from: 2011-12-22 Created: 2011-12-22 Last updated: 2012-04-03Bibliographically approved
2. Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells
Open this publication in new window or tab >>Transduction and oncolytic profile of a potent replication-competent adenovirus 11p vector (RCAd11pGFP) in colon carcinoma cells
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Keyword
gene-transfer vector; carcinoembryonic antigen; nucleotide-sequence; colorectal-cancer; cellular receptor; cd46 receptor; therapy; virus; expression; serotype-3
National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:umu:diva-41973 (URN)10.1371/journal.pone.0017532 (DOI)21455297 (PubMedID)
Available from: 2011-04-04 Created: 2011-04-04 Last updated: 2017-12-11Bibliographically approved
3. Replication-competent Ad11p vector (RCAd11p) efficiently transduces and replicates in hormone-refractory metastatic prostate cancer cells
Open this publication in new window or tab >>Replication-competent Ad11p vector (RCAd11p) efficiently transduces and replicates in hormone-refractory metastatic prostate cancer cells
Show others...
2009 (English)In: Human Gene Therapy, ISSN 1043-0342, E-ISSN 1557-7422, Vol. 20, no 4, 361-373 p.Article in journal (Refereed) Published
Abstract [en]

Selective replication-competent adenovirus serotype 5 vectors have been used for prostate cancer therapy. Unfortunately, gene transfer is inefficient because hormone-refractory metastatic prostate cancer cells have minimal coxsackievirus-adenovirus receptor expression. Vectors based on species B adenoviruses are attractive tools for use in human gene therapy because the viruses have low seroprevalence and they have efficient transduction capacity. Most species B adenoviruses use ubiquitously expressed complement-regulatory CD46 protein as a cellular receptor. Here we report the transduction efficacy and oncolytic capacity of a replication-competent Ad11p (RCAd11p) vector in human prostate cancer cells. Green fluorescent protein was efficiently expressed in a dose-dependent manner in PC-3 and DU 145 cells derived from metastasis of prostate cancer to bone and brain, respectively. However, transduction was less effective in LNCaP cells derived from prostate cancer metastasis to lymph nodes. The oncolytic capacity of the RCAd11p vector was 100 times higher in PC-3 cells than in the two other cell lines. The oncolysis was independent of the level of expression of p53 in the cells or on the absence of E1B55k expression in the vector. In vivo experiments revealed significant growth inhibition of PC-3 tumors in the xenograft mouse group treated with RCAd11p vector or Ad11pwt in comparison with the untreated control group. Thus, we have demonstrated that RCAd11p vector intrinsically possesses oncolytic properties, which were active in targeting tumor cells. Consequently, the novel RCAd11p vector has great potential for the treatment of incurable metastatic prostate disease.

Keyword
coxsackie adenovirus receptor; cellular attachment receptors; acute hemorrhagic cystitis; tumor-cells; gene-transfer; bladder-cancer; measles-virus; human cd46; in-vivo; p53
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-30381 (URN)10.1089/hum.2007.124 (DOI)19199789 (PubMedID)
Available from: 2009-12-19 Created: 2009-12-19 Last updated: 2017-12-12Bibliographically approved
4. Replication-competent adenovirus 11p GFP vector (RCAd11pGFP) enhances CEACAM6 expression in colon cancer cells
Open this publication in new window or tab >>Replication-competent adenovirus 11p GFP vector (RCAd11pGFP) enhances CEACAM6 expression in colon cancer cells
(English)Manuscript (preprint) (Other academic)
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-50808 (URN)
Available from: 2011-12-22 Created: 2011-12-22 Last updated: 2012-04-03Bibliographically approved

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