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Original Article
Cell Cycle Regulatory Protein Expression Profiles by Adenovirus p53 Infection in Human Papilloma Virus-associated Cervical Cancer Cells
Yong-Seok Lee, Su-Mi Bae, Sun-Young Kwak, Dong-Chun Park, Yong-Wook Kim, Soo-Young Hur, Eun-Kyung Park, Byoung-Don Han, Young-Joo Lee, Chong-Kook Kim, Do Kang Kim, Woong-Shick Ahn
Cancer Res Treat. 2006;38(3):168-177.   Published online June 30, 2006
DOI: https://doi.org/10.4143/crt.2006.38.3.168
AbstractAbstract PDFPubReaderePub
Purpose

The tumor suppressor gene, p53, has been established as an essential component for the suppression of tumor cell growth. In this study, we investigated the time-course anticancer effects of adenoviral p53 (Adp53) infection on human ovarian cancer cells to provide insight into the molecular-level understanding of the growth suppression mechanisms involved in Adp53-mediated apoptosis and cell cycle arrest.

Materials and Methods

Three human cervical cancer cell lines (SiHa, CaSki, HeLa and HT3) were used. The effect of Adp53 infection was studied via cell count assay, cell cycle analysis, FACS, Western blot and macroarray assay.

Results

Adp53 exerts a significant role in suppressing cervical cancer cell growth. Adp53 also showed growth inhibitory effects in each cell line, and it induced apoptosis and cell cycle arrest. Adp53 differentially regulated the expression of genes and proteins, and the gene expression profiles in the SiHa cells revealed that the p21, p53 and mdm2 expres sions were significantly up-regulated at 24 and 48 hr. Western blot shows that the p21 and p53 expression-levels were significantly increased after Adp53 infection. In addition, in all cell lines, both the CDK4 and PCNA protein expression levels were decreased 48 h after Adp53 infection. Cell cycle arrest at the G1 phase was induced only in the SiHa and HeLa cells, suggesting that exogenous infection of Adp53 in cancer cells was significantly different from the other HPV-associated cervical cancer cells.

Conclusion

Adp53 can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest, as well as through the regulation of the cell cycle-related proteins. The Adp53-mediated apoptosis can be employed as an advanced strategy for developing preferential tumor cell-specific delivery.

Citations

Citations to this article as recorded by  
  • Health‐promoting bioactivity and in vivo genotoxicity evaluation of a hemiepiphyte fig, Ficus dubia
    Uthaiwan Suttisansanee, Pornsiri Pitchakarn, Pisamai Ting, Woorawee Inthachat, Parunya Thiyajai, Daraphan Rodthayoy, Jirarat Karinchai, Bhanumas Chantarasuwan, Onanong Nuchuchua, Piya Temviriyanukul
    Food Science & Nutrition.2021; 9(4): 2269.     CrossRef
  • Comprehensive Data of P53 R282 Gene Mutation with Human Papillomaviruses (HPV)-Associated Oral Squamous Cell Carcinoma (OSCC)
    Tipaya Ekalaksananan, Weerayut Wongjampa, Pensiri Phusingha, Jureeporn Chuerduangphui, Patravoot Vatanasapt, Supannee Promthet, Natcha Patarapadungkit, Chamsai Pientong
    Pathology & Oncology Research.2020; 26(2): 1191.     CrossRef
  • Etoposide radiosensitizes p53-defective cholangiocarcinoma cell lines independent of their G2 checkpoint efficacies
    Arunee Hematulin, Sutiwan Meethang, Kitsana Utapom, Sopit Wongkham, Daniel Sagan
    Oncology Letters.2018;[Epub]     CrossRef
  • Effect and Safety of Recombinant Adenovirus-p53 Transfer Combined with Radiotherapy on Long-Term Survival of Locally Advanced Cervical Cancer
    Xing Su, Wen-juan Chen, Shao-wen Xiao, Xiao-fan Li, Gang Xu, Jian-ji Pan, Shan-wen Zhang
    Human Gene Therapy.2016; 27(12): 1008.     CrossRef
  • Celecoxib, a COX-2 Selective Inhibitor, Induces Cell Cycle Arrest at the G2/M Phase in HeLa Cervical Cancer Cells
    Agustina Setiawati
    Asian Pacific Journal of Cancer Prevention.2016; 17(4): 1655.     CrossRef
  • Cisplatin sensitivity and mechanisms of anti-HPV16 E6-ribozyme on cervical carcinoma CaSKi cell line
    Zhiguo Rao, Jianfei Gao, Bicheng Zhang, Bo Yang, Jiren Zhang
    The Chinese-German Journal of Clinical Oncology.2012; 11(4): 237.     CrossRef
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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
AbstractAbstract PDF
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
AbstractAbstract 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
  • 3,945 View
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Cell-Specific Growth Inhibition of Human Cervical Cancer Cell by Recombinant Adenovirus p53 in vitro and in vivo
Su Mi Bae, Yong Wook Kim, Joo Hee Yoon, Jin Young Yoo, Young Seok Seo, Sang Lyun Nam, Joon Mo Lee, Sung Eun Namkoong, Chong Kook Kim, Yong Wan Kim, Woong Shick Ahn
Cancer Res Treat. 2003;35(3):181-190.   Published online June 30, 2003
DOI: https://doi.org/10.4143/crt.2003.35.3.181
AbstractAbstract PDF
PURPOSE
Despite the significance of the p53 adenoviral vector in cancer gene therapy, an advanced strategy for the development of preferential tumor cell-specific delivery and the long-term persistent gene expression control of p53 are required. In this study, the time-course expression patterns of p53 and E6, on cervical cancer cells, were investigated to obtain a molecular level understanding of the cell-dependent tumor growth suppression effects of a recombinant adenovirus expressing p53, both in vitro and in vivo. MATERIALS AND METHODS: The expressions of p53 and E6 in CaSki, SiHa, HeLa, HeLaS3, C33A and HT3 cervical cancer cell lines were examined. After infection with AdCMVp53, the cell growth inhibition was studied via cell count, MTT and Neutral red assays. After transfecting the AdCMVp53 and AdCMVLacZ into the cancer cells-xenografted nude mice, the anti-tumor effects were investigated for one month. RESULTS: The p53 protein levels were more notably expressed in the CaSki and HeLa than in the SiHa and HeLaS3 On day 6, the p53 was only detected in the HeLaS3. In contrast, the p53 expression was highly maintained in the C33A and HT3. The E6 mRNA levels gradually decreased in only the CaSki and HeLa. The growth suppression effects also showed cell-dependent patterns, which were consistent with the reciprocal expression patterns of p53 and E6. After transfection of the AdCMVp53, into the CaSki- and SiHa-xenografted nude mice, the tumor size was remarkably decreased in the SiHa cells as compared to that in the AdCMVLacZ transfected mice, indicating cell-specific growth inhibition patterns.
CONCLUSION
The adenovirus-mediated p53 gene transfection was very effective both in vitro and in vivo. Also, the anti-tumor effects were accomplished via the differential role of p53-specific apoptotic cell death, which was dependent on the cervical cancer cell line.

Citations

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  • Immunization with Adenoviral Vectors Carrying Recombinant IL-12 and E7 Enhanced the Antitumor Immunity against Human Papillomavirus 16-associated Tumor
    Eun-Kyung Park, Young-Wook Kim, Joon-Mo Lee, Sung-Eun NamKoong, Do-Gang Kim, Heung-Jae Chun, Byoung-Don Han, Su-Mi Bae, Hyun-Sun Jin, Jeong-Im Sin, Woong-Shick Ahn
    Cancer Research and Treatment.2005; 37(1): 63.     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
AbstractAbstract 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.
AbstractAbstract 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.
AbstractAbstract 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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Induced Apoptosis of Cancer Cells by Antisense ATM Gene Therapy
H K Yi, S H Chang, D Y Lee, J S Kim, R H Hwang
J Korean Cancer Assoc. 2000;32(2):339-349.
AbstractAbstract PDF
PURPOSE
Recently cloned AT gene mutated (ATM) in ataxia telangiectasia is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wide role in signal transduction. ATM phenotype cells increased sensitivity to ionizing radiation and chemother- apeutic agents, and are defective Gl/S checkpoints in response to DNA damage. We have trying to focus on the possible role of hypersensitivity to DNA damage-induced apoptosis in the ATM cells. We hypothesized that cancers could be made more sensitive to therapeutic x-rays and agents by disabling the AT gene in tumor cells using ATM antisense strategy. Therefore, we investigated whether antisense ATM transduced cancer cells show ATM phenotype cells and undergo apoptotic death after exposure to low concentration of chemotherapeutic agents or irradiation doses that do not induce appreciable apoptosis in control cells.
MATERIALS AND METHODS
NH2-terminal portion of AT gene was recloned and ligated in reverse orientation in pcDNA3. This antisense ATM expression vector transfected to MCF-7 cells, and Ve3 cells by calcium phosphate method. The transformed cells were selected in the presence of G418 and confirmed AT protein expression by immunohistochemistry. We have analyzed the induction of apoptosis of antisense ATM transduced MCF-7 and Ve3 cells after treatment of chemotherapeutic agents or irradiation using DNA fragmentation, MTT assay and FACScan.
RESULTS
Antisense ATM transduced Ve3 and MCF-7 cells were showed the inhibition of ATM protein expression. Its expression was predominantly detected in the nucleus. The antisense ATM transduced Ve3 and MCF-7 cells showed increased progress of DNA fragmentaion induced by etoposide treatment, increased radiosensitization and induced apototic cells (Ao phase) of FACScan after irradiation, and increased susceptibility of apoptosis induced by treatment with low concentration of chemotherapeutic agents.
CONCLUSIONS
ATM playing in cell cycle progression and cell death in response to DNA damage may help identify potential targets for improved cancer therapies, and ATM antisense gene therapy can be a useful therapeutic tool to combat various cancers.
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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.
AbstractAbstract 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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Restoration of Hormone Dependency in Estrogen Receptor - Lipofected MDA-MB-231 Human Breast Cancer Cells
Young Jin Suh, Jae Hee Chang, Chung Soo Chun
J Korean Cancer Assoc. 1999;31(3):473-482.
AbstractAbstract PDF
PURPOSE
The loss of estrogen and progesterone receptors appeats to be associated with a progression to less differentiated and hormone-independent tumors. The gain of hormone independency over time even in estrogen receptor-positive tumors has become another obstacle to endocrine therapy for breast cancer. We tried to regain the hormone dependency in estrogen receptor-negative breast cancer cells by lipofecting estmgen receptor cDNA.
MATERIALS AND METHODS
The mutant human estrogen receptor cDNA (pSGS-HEO) was lipofected into estrogen receptor-negative human breast cancer cell line MDA-MB-231, in an attempt to restore their sensitivity to antiestrogen. Then the effects of 17p-estradiol and tamoxifen were studied by counting viable cell numbers after treating the lipofected cell line with either one or together.
RESULTS
Culture medium cantaining phenol red, a weak estrogen, has growth advantages compared with culture medium without it. In both culture conditions, cell growth was most profoundly inhibited in 4 days after lipofection with mutant human estrogen receptor cDNA, which was overcome after that day. Tamoxifen, as an antiestrogen, showed a growth inhibitory effect slightly stronger tban combined conditions of tamoxifen and 17- estradiol compared to estrogen-treated group and to control, and the inhibitory effect was lasted 4 days.
CONCLUSION
The temporary induction of estrogen receptor by lipofection with pSGS-HEO on estrogen receptor-negative human breast cancer cell line MDA-MB-231 showed negative growth control on these cells by tamoxifen, indicating that liposome-mediated estrogen receptor transfection may be used as a novel therapeutic strategy for hormane independent human breast cancers in the near future.
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Effects of Herpes Simplex Virus - Thymidine Kinase Gene Transduction into the Hepatocellular Carcinoma Cell Lines Using the Retrovirus on Ganciclovir Cytoxicity
Joo Hang Kim, Jae Jin Song, Yoon Soo Chang, Eun Hee Kim, Jae Sung Kim, Heui Ran Lee, Jae Kyung Roh, Byung Soo Kim, Joong Bae Ahn, Nae Chun Yoo, Hyun Cheol Chung
J Korean Cancer Assoc. 1998;30(5):1034-1043.
AbstractAbstract PDF
PURPOSE
Hepatocellular carcinoma (HCC) is one of the most common malignancy with high mortality in Korea. A new therapeutic modality such as gene therapy is necessary to improve the prognosis of hepatoma patients. Therefore we investigated the preclinical significance of Herpes simplex virus - thymidine kinase/ganciclovir (HSV-tk/GCV) gene therapy model using the retroviral vector for HCC cell lines.
MATERIALS AND METHODS
LNC/HSV-tk retroviral vector and PA317/LNC/HSV-tk pro- ducer cell line were constructed. HSV-tk transduced HCC cells using the LNC/HSV-tk retrovirus were selected by the G418 containing media. In vitro GCV sensitivity test of the HCC cells was performed by MTT assay. To evaluate in vivo GCV sensitivity, GCV was intraperitoneally injected after subcutaneous administration of HCC cells into each flank of the nude mouse.
RESULTS
HSV-tk gene transduction and expression in HCC cells were confirmed by RT-PCR. HSV-tk transduced HCC cell lines (SK-Hepl/HSV-tk and Hep-3B/HSV-tk) showed the marked GCV sensitivity comparing with the parental cell lines (SK-Hepl and Hep-3B) by MTT assay (p<0.001). The MTT test revealed that SK-Hepl/HSV-tk cells were more sensitive to GCV compare with that of Hep-3B/H5V-tk cells, and the parent cell line showed minimal growth suppression by the GCV treatment. In 12 nude mice received tumor cell mixtures of Hep-3B and Hep-3B/HSV-tk cells which contained more than 50% of HSV-tk transduced cells, the tumor was not developed in ll mice by the intraperitoneal administration of GCV. The tumors developed in 1 of 6 mice and 5 of 6 mice when mixtures contained 30% and 10% of HSV-tk transduced cells, respectively. Five mice out of 6 mice received inoculum containing the mixtures of 70% and 50% of HSV-tk transduced cells into each flank survived more than 6 month after HSV-tk/GCV treatment. Conelusion: HSV-tk gene transduced HCC cells showed the enhanced sensitivity to GCV. In nude mice HSV-tk/GCV strategy for HCC seemed to be more effective when tumor cell inoculum contained more than 30% of HSV-tk transduced HCC cells.
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Gene Transfer Effects of Thymidine Kinase Gene of Herpes Simplex Type 1 on Ganciclovir Cytotoxicity in Gastric Cancer Cell Line
Jae Kyung Roh, Soo Jung Gong, Joo Hang Kim, Hyo Dong Um, Nae Chun Yoo, Jin Hyuk Choi, Jae Jin Song, Sun Young Rha, Hyun Cheol Chung, Jin Sik Min, Byung Soo Kim
J Korean Cancer Assoc. 1998;30(1):20-30.
AbstractAbstract PDF
PURPOSE
Gastric cancer is the most common malignancy in Korea. Although treatment such as surgery, chemotherapy, and immunotherapy has greatly improved, the mortality rate of gastic cancer is still high, A new therapeutic trial is necessary to improve the cure rate of gastric cancer. Therefore we investigated the pre-clinical significance of HSV-tk gene therapy using retroviral vector for gastric cancer cell lines.
MATERIALS AND METHODS
LNC/HSV-tk retroviral vector and PA317/LNC/HSV-tk producer cell line were constructed. HSV-tk gene transduction and expression were detected by PCR. An in vitro ganciclovir(GCV) sensitivity test was performed by MTT assay. To evaluate in vivo GCV sensitivity, GCV was intraperitoneally injected after tumor formation in the nude mice. Bystander effect was observed in vitro MTT assay using YCC- S-2 cell line and in vivo using N87 and YCC-S-2 cell lines.
RESULTS
The in vitro GCV sensitivity test showed that the growth inhibition was 30~32% with 0.5 uM GCV and 52~77% with 500 uM GCV in the HSV-tk transduced cell line in comparison with 0- 5% with 0.5 and 500 uM GCV in the parent cell line. The in vivo GCV administration showed that the tumors induced by HSV-tk transduced N87 cell line and YCC-S-2 cell line decreased completely, while the tumors with the parent cell lines continued to grow in nude mice. We observed no tumor cells in tissue specimen of the tumor induced by the N87/HSV-tk cell line after. GCV administration. In vitro and in vivo bystander effects were observed in HSV-tk/GCV system due to the resultant cell death exceeding the proportion of HSV-tk transduced cells in the mixtures of HSV-tk transduced and parent cells.
CONCLUSION
HSV-tk transduced gastric cancer cell lines showed sensitivity to GCV and a bystander effect was observed. These results suggested that HSV-tk/GCV system should be evaluated in the clinical settings.
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A Study of Retrovirus-mediated p53 Gene Transduction Into Human Gastric Cancer Cell Lines
Joo Hang Kim, Yoo Sun Moon, Dong Hwan Shin, Jae Jin Song, Soo Jung Gong, Sun Young Rha, Soo Kyoung Kim, Sook Jung Jeong, Hyun Cheol Chung, Jae Kyung Roh, Jin Sik Min, Byung Soo Kim
J Korean Cancer Assoc. 1997;29(5):754-764.
AbstractAbstract PDF
PURPOSE
The development of new therapeutic modalities such as gene therapy, which still requires further investigation, is clearly important to improve the prognosis of gastric cancer. This study was conducted to evaluate the effect on the growth and the tumorigenicity of retrovirus-mediated p53 gene transduction into gastric cancer cells.
MATERIALS AND METHODS
Human gastric cancer cell lines were cultured and their DNAs were analyzed to evaluate the p53 status with PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) and DNA sequencing. Retroviral supernatants were obtained from each producer cell line, PA317/LNCX and PA317/LNC/p53, after construction of retroviral vector LNC/p53 containing human p53 cDNA and producer cell line PA 317/ LNC/p53. To investigate the effect of retrovirus-mediated p53 gene transduction in human gastric cancer cell lines, the in vitro growth rates and in vivo tumorigenicities of the N-87 cell line having mutant p53 and the YCC-S-2 cell line having wild-type p53 were compared before and after infection with LNC/p53 retrovirus. RESULTS: The following results were obtained: 1) The growth inhibition of N-87 cells after p53 transduction was signficant when compared to that of the parent N-87 cells. The growth of the p53 transduced YCC-S-2 cells and the parent YCC-S-2 cells was not different. 2) In nude mice, the growth of tumors formed by N-87 cells was modestly inhibited after retrovirus-mediated wild-type p53 gene transduction. However, the growth of tumors formed by YCC-S-2 cells was not inhibited by retrovirus-mediated p53 gene transduction. 3) The expression rate of p53 protein after p53-containing retroviral infection in the KATO-III cell lines, which have no p53 gene, was dose-dependent on the m.o.i. of retrovirus, although it was not more than 15% with the m.o.i. of 100 upon immunohistochemical analysis.
CONCLUSION
The growth inhibition by retrovirus-mediated p53 transduction in human gastric cancer cells was significant in a gastric cancer cell line having mutant p53 in vitro, and the growth of tumor masses formed by a gastric cancer cell line having mutant p53 was modestly inhibited after p53 transduction using retroviral vector in nude mice, although it was not statistically significant. Only modest inhibition of tumor growth using retrovirus-mediated p53 gene transduction in vivo is most likely to be due to low transduction efficiency.
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Combined Therapy of Gene Therapy using the Herpes Simplex Virus Thymidine Kinase, and Retinoic Acid
Jae Yong Park, Steven M Albelda
J Korean Cancer Assoc. 1997;29(2):321-331.
AbstractAbstract PDF
PURPOSE
Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk/GCV) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin expression, we hypothesized that these compound could be used to augment the HSVtk/GCV bystander effect.
MATERIALS AND METHODS
We transferred HSVtk gene to AB12 cell line that express connexin43 as a component of gap junction. We examined the effects of retinoic acid (RA) on GJIC utilizing a functional double-dye transfer study. To evaluate the bystander effect in vivo, a murine subcutaneous tumor model was established. Before proceeding with comparisons of HSVtk/GCV mediated bystander cell killing, we evaluated the effects of RA on flank tumor growth in order to rule out a potential antitumor effect of RA alone. Then we determined the effects of retinoic acid on bystander-mediated cell killing in an animal model.
RESULTS
Addition of all-trans retinoic acid increased GJIC in AB12 cell line and was associated with more efficient GCV induced bystander killing in animal model. HSVtk transduced tumors in mice treated with the combination of GCV and retinoids were significantly smaller than those treated with GCV or retinoids alone.
CONCLUSION
These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effects and may thus be a promising new approach to improve response in gene therapy utilizing the HSVtk/GCV system to treat tumors.
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Comparison of Efficiency of Infection of Human Cancer Cell Lines Via Retroviral Vector System
Yeon Soo Kim, Hee Kyung Lim, Joo Hang Kim, Jin Sik Min
J Korean Cancer Assoc. 1997;29(1):1-10.
AbstractAbstract PDF
PURPOSE
There are several reasons why retroviruses are useful as vectors for gene therapy. However, retroviral vectors also have some limitations. Research in retroviral-mediated gene transfer has struggled with low titer and transduction efficiency on certain human target cells even with the addition of polycations to enhance transduction. Efficient in vivo gene transfer with retroviral vectors will require the availability of large amounts of vector at titers higher than generally possible by most current methods. Therefore, transduction efficiency of various human cell types with retroviral vector system is very important in human gene therapy. In an effort to test the transduction efficiency of a retrovial vector in the human cancer cell lines, a retroviral vector was infected into various human cancer cell lines.
MATERIALS AND METHODS
We generated retrovirus producing cell lines through transfection or infection of amphotropic packaging cell line PA317 with ecotropic retroviruses encoding bacterial lacZ gene. The amphotropic retrovirus vector was used to transduce various human cancer cell lines.
RESULTS
Of eight randomly chosen G418-resistant clones generated by transfection, only two clone produced the vector at up to >10 (6) cfu/ml, while one of five clones generated by infection yielded higher-titer virus in the absence of helper virus, up to 1 X 10 (7) cfu/ml, than the transfected clones. Transduction with supernatant derived from a PA317 producer cell line has resulted in transduction levels from 1% to 15%, 5- to 60-fold lower than those analyzed in NIH3T3 cells.
CONCLUSION
These findings suggest that new improved gene transfer method into human cancer cells using retroviruses is required for efficient in vivo cancer gene therapy.
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Retroviral Vector - mediated Tumor Necrosis Factor - α Gene Transfer into Human Gastric Carcinoma Cell Lines
Jung Ae Rhee, Jung Ae Lee, Dae Seog Heo, Sung Koo Han, Noe Kyeong Kim
J Korean Cancer Assoc. 1994;26(5):677-688.
AbstractAbstract PDF
The tumor necrosis factor a (TNF-a) is a potent cytokine with antitumor activities including a direct cytotoxic effect on human cancer cells and the enhancement of a cytotoxic immune response against the tumor. However, its effectiveness in the human clinical trials is limited due to severe systemic toxicities. An alternative approach, that tumor cells are genetically engineered to secrete TNF-a locally to stimulate the immune system without systemic toxicities, is suggested as a form of gene therapy (tumor-cell-targeted lymphokine gene therapy). In this trial, cDNA encoding human TNF-a (TNF-NeoR) was introduced into five human gastric carcinoma cell lines using a retroviral vector to examine whether TNF-a gene could be transfected and expressed in gastric carcinoma cell lines in vitro. Successful transfer of TNF-a gene into five gastric csrcinoma cell lines was confirmed by polymerase chain reaction techniques. Supernatants (1: 2 dilution) from cultures of transduced gastric carcinoma cell lines demonstrated cytotoxicity to TNF-sensitive WEHI 164 cell lines in the range of 20-49%. TNF- transduced gastric carcinoma cell lines secreted TNF-a at the concetration of 479-8869 pg/10(6) cells-24 hours, whereas the parental cell lines did not secrete TNF-a. There were no differences in the growth rates between parental and TNF-transduced cell lines in vitro. The four TNF-transduced SNU cell lines showed the resistance to endogenous and exogenous TNF, except SNU-668 cell line. In conclusion, TNF-a gene was successfully transfected and expressed in gastric carcinoma cell lines in vitro. These data will be helpful in the development of tumor-cell-targeted lymphokine gene therapy for the treatment of advanced gastric carcinoma.
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An Antibody Against E2 / MIC2 Antigen ( CD99 ) : Indentification and Characterization
Kyeong Cheon Jung, Tae Jin Kim, Doo Hyun Chung, Kuhn Kuk Lee, Jang Hee Hahm, Seong Hoe Park
J Korean Cancer Assoc. 1996;28(2):350-358.
AbstractAbstract PDF
Liposome-mediated gene transfer offers the potential to introduce DNA encoding therapeutic DNA to treat human disease. Several genes have been used in gene therapy to stimulate immune response in malignancy, and allogeneic major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. This study was performed to determine the transfection efficiency and safety of liposome- mediated gene delivery into human cancer cell lines and animals. Placental alkaline phosphatase gene as a reporter was transfected into PCI-13 and NCI-H522 cell lines, and the expression was determined in individual transfected cells by histochemical staining. The transfectian efficiency was the highest at 24 ¥ig of DNA mixed with 10¥il of lipofectamine in vitro, HLA-B7 DNA as a therapeutic gene was transfected into cell lines and injected subcutaneously into the rabbits. The HLA-B7 gene expression was successfully identified by RT-PCR in vivo and in vitro. Twenty percents of the cells expressed HLA-B7 proteins which were analyzed by flow cytometry. In rabbit model, no pyrogenic response was ob- served after subcutaneous injection of HLA-B7/liposome complexes. The expression of injected HLA-B7 gene was restricted within the injected skin. These data showed the feasibility and safety of liposome-mediated gene transfer. These findings will provide a basis to develop strategies for the gene therapy of human cancer.
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Preclinical Study of HLA-B7 DNA / Liposome Complex for Gene Therapy
Seong Jun Yoon, Won Seok Kim, Jae Go Seol, Sagn Goo Lee, Kee Hyung Lee, Dae Seog Heo, Noe Kyeong Kim
J Korean Cancer Assoc. 1996;28(2):358-368.
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