Cancer Research and Treatment

Original Article

Increased Cytopathic Effect of Replicating Adenovirus Expressing Adenovirus Death Protein: Eunhee Kim, Joo Hang Kim, Taeyoung Koo, Joo Hyuk Sohn, Chae Ok Yun; Cancer Res Treat. 2003;35(5):425-432. Published online October 31, 2003; DOI: https://doi.org/10.4143/crt.2003.35.5.425

PURPOSE
Replication-competent adenoviruses (Ads) are promising new modalities for the treatment of cancer. Selective replication of a viral agent in tumor may lead to improved efficacy over non-replicating Ads due to viral multiplication, lysis of the infected cancer cell and spread to surrounding cells. In our previous studies it was shown that the E1B 55 kD-deleted Ad (YKL-1) exhibits tumor specific replication and cell lysis, but with reduced cytolytic effects compared to the wild type adenovirus (Int J Cancer 2000;88: 454-463). Thus, improving the potency of oncolytic Ads remains an important goal for cancer gene therapy. To increase the oncolytic ability of YKL-1, an adenovirus death protein (ADP) gene was reintroduced under the control of a CMV or MLP promoter at the E3 region of the YKL-1, generating an YKL-cADP and YKL-mADP, respectively.
MATERIALS AND METHODS
The in vitro cytolytic effect of ADP expressing Ads was evaluated by MTT assay, and the induction of apoptosis by ADP expressing Ads was examined by TUNEL analysis. Finally, the antitumor effect of ADP expressing Ads was demonstrated in C33A xenograft tumor model. RESULTS: The YKL-cADP exerted a markedly enhanced cytolytic effect against H460 and SK-Hep1 cancer cell lines. The TUNEL assay indicated that the ADP-mediated cytotoxicity was largely driven by apoptosis. Finally, the YKL-cADP showed a superior antitumor effect than the YKL-1 or YKL-mADP in C33A xenografts. CONCLUSION: These lines of evidence demonstrate that the YKL-cADP induces efficient cell lysis, which is critical for the addition of therapeutic value to replicating Ads in cancer gene therapy.

Citations

Citations to this article as recorded by

The Adenovirus Death Protein – a small membrane protein controls cell lysis and disease
Fanny Georgi, Urs F. Greber
FEBS Letters.2020; 594(12): 1861. CrossRef
A compendium of adenovirus genetic modifications for enhanced replication, oncolysis, and tumor immunosurveillance in cancer therapy
Aleksei A. Stepanenko, Vladimir P. Chekhonin
Gene.2018; 679: 11. CrossRef
Oncolytic viruses: adenoviruses
Julia Niemann, Florian Kühnel
Virus Genes.2017; 53(5): 700. CrossRef

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Original Articles

Development of a Conditional Replication Competent Adenovirus, Controlled by the Human Telomerase Promoter (hTERT): Eunhee Kim, Joo Hang Kim, Ha Youn Shin, Han Saem Lee, Joo Hyuk Sohn, Jai Myung Yang, Jungho Kim, Chae Ok Yun; Cancer Res Treat. 2003;35(3):191-206. Published online June 30, 2003; DOI: https://doi.org/10.4143/crt.2003.35.3.191

Abstract PDF

PURPOSE
This study has been planned to generate a replication-competent adenovirus which replicates in a cancer cell-specific manner, thus minimizing the side effects and toxicity of cancer gene therapy. MATERIALS AND METHODS: we have generated an E1B 19 kD attenuated recombinant adenoviruses, Ad-TERT-delta19 and Ad-mTERT-delta19, which encode E1A gene driven by the wild type hTERT and modified m-hTERT promoter containing additional c-myc and Sp1 binding sites in the backbone of Ad-deltaE1B19. The in vitro efficacy and specificity of the hTERT and m-hTERT promoter have been evaluated by the comparison of viral replication and cytopathic effect in cancer cells and normal cell lines. To assess anti-tumor effect and safety of hTERT or m-hTERT promoter driven replication competent adenoviruses, tumor regression after subcutaneous injection in subcutaneous C33A xenografts and lacZ expression after systemic injection in organs were examined. RESULTS: The activation of hTERT or m-hTERT promoter was significantly up-regulated only in hTERT-positive cells, but not in hTERT-negative cells. Moreover, the activity of m-hTERT promoter was substantially increased in hTERT-positive cancer cells, but not in hTERT-negative cells. While Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect in cancer cells only. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. CONCLUSIONS: The use of m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of m-hTERT promoter may be a new promising tool for the treatment of human malignancies.

Citations

Citations to this article as recorded by

Bone and Soft-Tissue Sarcoma: A New Target for Telomerase-Specific Oncolytic Virotherapy
Hiroshi Tazawa, Joe Hasei, Shuya Yano, Shunsuke Kagawa, Toshifumi Ozaki, Toshiyoshi Fujiwara
Cancers.2020; 12(2): 478. CrossRef
Impact of Autophagy in Oncolytic Adenoviral Therapy for Cancer
Hiroshi Tazawa, Shinji Kuroda, Joe Hasei, Shunsuke Kagawa, Toshiyoshi Fujiwara
International Journal of Molecular Sciences.2017; 18(7): 1479. CrossRef

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Evaluation of E1B-mutant Replicating Adenoviruses for Cancer Gene Therapy: Jae Sung Kim, Joo Hang Kim, Heui Ran Lee, Kyeong Cheon Jung, Chae Ok Yun; Cancer Res Treat. 2001;33(6):500-511. Published online December 31, 2001; DOI: https://doi.org/10.4143/crt.2001.33.6.500

Abstract PDF: PURPOSE
Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. In an effort to continually improve upon cancer gene therapy, we have modified gene- attenuated replication-competent adenoviruses so as to cause them to replicate efficiently and lyse the infected cancer cells more effectively.
MATERIALS AND METHODS
We modified the E1 region of the adenovirus (Ad) systematically, generating Ad-deltaE1B19, Ad-deltaE1B55, Ad-deltaE1B19/55, and Ad-WT. The cytopathic effects (CPE) and viral replication of these four gene modified adenoviruses were compared, and the morphology and DNA fragmentation of the infected cells was evaluated.
RESULTS
Among the constructed adenoviruses, E1B 19kD-inactivated adenovirus (Ad-deltaE1B19) was the most potent, inducing the largest-sized plaques and markedCPE. Moreover, cells infected with Ad-deltaE1B19 showed complete cell lysis with disintegrated cellular structure whereas cells infected with Ad-WT maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL assay was also used to monitor DNA integrity, and a more profound induction of apoptosis was observed in the Ad-deltaE1B19 infected cells in comparison to wild type adenovirus infected cells.
CONCLUSION
We demonstrate that the inactivation of the E1B19kD gene in a replicating adenovirus leads to increased CPE, rapid viral release, improved cell-to-cell viral spread and increased induction of apoptosis.

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Antiangiogenesis Gene Therapy Using Adenovirus-mediated Antisense-VEGF in Glioblastoma Multiforme: Seock Ah Im, Jeong Soo Kim, Eunmi Nam, Soon Nam Lee; J Korean Cancer Assoc. 2000;32(4):764-774.

Abstract PDF: PURPOSE
Vascular endothelial growth factor (VEGF) is a major positive effector of angiogenesis. We investigated the mechanism of tumor growth inhibition by adenoviral transfer of antisense- VEGF in glioma and the role of VEGF for in vivo growth of human glioma cells according to the stage of the tumor growth.
MATERIALS AND METHODS
Replication-deficient adenoviral vector containing the VEGF cDNA in an antisense orientation (Ad5CMV-alphaVEGF) were constructed to increase the in vivo applicability of antisense sequence. The effect of Ad5CMV-alphaVEGF was studied in vitro and in vivo with human glioma cell line U-87 MG. Immunohistochemical staining of the subcutaneous tumor with anti-VEGF antibody and CD34 antibody were performed to compare VEGF protein expression and the microvessel count respectively.
RESULTS
The growth curve of U-87 MG cells treated with Ad5CMV-alphaVEGF remained as same as that of mock-infected and Ad5(dl312)-infected U-87 MG cells in vitro, suggesting that Ad5CMV-alphaVEGF does not have direct cytotoxic effect. The growth of subcutaneous human glioma xenografts was inhibited by early intratumoral injection of Ad5CMV-alphaVEGF. Immuno histochemical staining of tumors showed that VEGF protein expression and mean microvessel counts were decreased in early Ad5CMV-alphaVEGF treatment group.
CONCLUSION
The efficient down-regulation of VEGF produced by tumor cells using Ad5CMV- alphaVEGF in early stage of glioma growth has an antitumor effect in vivo through antiangiogenic mechanism.

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Effects of Integrin avB5 and av B53 on the Adenovirus - Mediated Gene Transduction: J Y Park, S Y Joo, H S Jeon, H J Chang, S Kam, I S Kim; J Korean Cancer Assoc. 2000;32(2):422-430.

Abstract PDF: PURPOSE
Human adenovirus, commonly used as a vector for gene therapy, enters host cells by receptor mediated endocytosis. Since integrin av B5 of cell surface promotes endocytosis of adenovirus and subsequent disruption of endosome, we hypothesized that the level of integrin av B5 of target cells may determine the efficiency of adenovirus- mediated gene transfer and its therapeutic effects.
MATERIALS AND METHODS
We transduced lacZ gene or herpes simplex virus thymidine kinase (HSVtk) gene, using adenoviral vector, into human lung cancer cell lines (H1299, H157, and H322). Then we evaluated the relationship between the level of integrin av B5 and av 53 of target cells, and adenovirus-mediated gene transduction efficiency.
RESULTS
The trasduction efficiency, observed by X-gal stain and B-gal activity after infection of recombinant-adenovirus encoding lacZ gene, was correlated with the level of integrin av B5, assessed by Western blotting. The bystander mediated cell killing, after transduction of HSVtk gene, was also correlated with the level of integrin av B5 of cell lines.
CONCLUSION
These results suggest that quantitative measurement of the level of integrin av B5 of target cells may be a useful predictor of the efficiency and effectiveness of gene transfer by means of an adenoviral vector.

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Tumor - specific Virus Replication and Cytotoxicity of E1B 55 kD - deleted Adenovirus: Jaesung Kim, Boyoung Lee, Jinahn Kim, Joong Bae Ahn, Joon Oh Park, Nae Chun Yoo, Joo Hang Kim, Jae Kyung Roh, Jin Sik Min, Byung Soo Kim, Heuiran Lee; J Korean Cancer Assoc. 2000;32(1):200-209.

Abstract PDF: PURPOSE
To overcome the limitations of cancer gene therapy using replication-incom- petent adenovirus, we generated E1B 55 kD-deleted adenovirus (YKL-1) by polymerase chain reaction (PCR) and homologous recombination. We then investigated tumor-specific virus replication and cytotoxicity of YKL-1 in vitro and in vivo.
MATERIALS AND METHODS
YKL-1 was constructed by reintroducting E1A and E1B 19 kD into pTG-CMV El/E3-deficient adenoviral vector and inducing homologous recombination in E. coli. The recombinant vector pYKL-1 was transfected into 293 cells to generate YKL-1. The properties of newly constructed YKL-1 was defined by PCR and immuno- blotting analysis. Virus replication was examined by infecting human normal and cancer cells on 6-wells at multiplicity of infection (MOI) of 10 for 3 days. Virus was then recovered and titered. Cytopathic effect was analyzed by infecting human normal and cancer cells on 24-wells at MOIs of 10, 1 or 0.1 for 7 to 10 days and staining them with crystal violet solution. Inhibition of tumor growth was examined in human cancer cell xenografts in nu/nu mice by intratumoral injection of YKL-l.
RESULTS
PCR and immunoblotting analysis confirmed that YKL-1 contained E1A and E1B 19 kD but not E1B 55 kD. In human normal cells, virus replication and subsequent cytopathic effect of E1B 55 kD-deleted adenovirus YKL-1 was markedly attenuated by larger than 2 to 3 log in magnitude, compared to that of wild-type ad-XJ. In contrast, YKL-1 was capable of replicating and inducing cytotoxicity i.n most human cancer cells. C33A and Hep3B containing p53 mutation were much more sensitive, whereas HeLa and H460 with wild type p53 were relatively resistant to YKL-1. Finally, the tumor growth was dramatically retarded by intratumoral injection of YKL-1 in C33A cervical cancer xenograft and the histology showed significant necrosis by intratumoral injection of YKL-1.
CONCLUSION
The results here demonstrated the ability of preferential virus replication and cytotoxicity of ElB 55 kD-deleted adenovirus YKL-1 in human cancer cells. Therefore, these indicated a promising potential of YKL-1 as an antitumoral virus agent and a selective replication-competent virus vector.

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