Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5- fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model

Toshiro Shirakawa, Thomas A. Gardner, Song Chu Ko, Neil Bander, Savio Woo, Akinobu Gotoh, Sadao Kamidono, Leland W K Chung, Chinghai Kao

Research output: Contribution to journalArticle

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Abstract

Purpose: An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods: Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results: In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV- TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad- RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TK/ACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion: We have demonstrated in this study that Ad-RSV- CD/5-FC is superior to Ad-RSVTK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.

Original languageEnglish
Pages (from-to)949-954
Number of pages6
JournalJournal of Urology
Volume162
Issue number3 I
DOIs
StatePublished - Sep 1999

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Cytosine Deaminase
Flucytosine
Acyclovir
Thymidine Kinase
Renal Cell Carcinoma
Genetic Therapy
Connexin 43
Gap Junctions
Electron Microscopy
Western Blotting
Rous sarcoma virus
Cell Line
Neoplasms
Intercellular Junctions
Poisons
Virus Diseases
Growth
Radiotherapy
Animal Models
Cell Culture Techniques

Keywords

  • Animal model
  • Experimental therapeutics
  • Human kidney cancer

ASJC Scopus subject areas

  • Urology

Cite this

Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5- fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model. / Shirakawa, Toshiro; Gardner, Thomas A.; Ko, Song Chu; Bander, Neil; Woo, Savio; Gotoh, Akinobu; Kamidono, Sadao; Chung, Leland W K; Kao, Chinghai.

In: Journal of Urology, Vol. 162, No. 3 I, 09.1999, p. 949-954.

Research output: Contribution to journalArticle

Shirakawa, Toshiro ; Gardner, Thomas A. ; Ko, Song Chu ; Bander, Neil ; Woo, Savio ; Gotoh, Akinobu ; Kamidono, Sadao ; Chung, Leland W K ; Kao, Chinghai. / Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5- fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model. In: Journal of Urology. 1999 ; Vol. 162, No. 3 I. pp. 949-954.
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abstract = "Purpose: An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods: Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results: In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV- TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad- RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TK/ACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion: We have demonstrated in this study that Ad-RSV- CD/5-FC is superior to Ad-RSVTK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.",
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author = "Toshiro Shirakawa and Gardner, {Thomas A.} and Ko, {Song Chu} and Neil Bander and Savio Woo and Akinobu Gotoh and Sadao Kamidono and Chung, {Leland W K} and Chinghai Kao",
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T1 - Cytotoxicity of adenoviral-mediated cytosine deaminase plus 5- fluorocytosine gene therapy is superior to thymidine kinase plus acyclovir in a human renal cell carcinoma model

AU - Shirakawa, Toshiro

AU - Gardner, Thomas A.

AU - Ko, Song Chu

AU - Bander, Neil

AU - Woo, Savio

AU - Gotoh, Akinobu

AU - Kamidono, Sadao

AU - Chung, Leland W K

AU - Kao, Chinghai

PY - 1999/9

Y1 - 1999/9

N2 - Purpose: An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods: Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results: In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV- TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad- RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TK/ACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion: We have demonstrated in this study that Ad-RSV- CD/5-FC is superior to Ad-RSVTK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.

AB - Purpose: An estimated 11,600 Americans will die of renal cell carcinoma in 1998. The lack of effective chemotherapy or radiotherapy requires the investigation of novel treatment modalities. We compared two forms of toxic gene therapy, cytosine deaminase (CD) plus 5-fluorocytosine (5-FC) and thymidine kinase (TK) plus acyclovir (ACV), in pre-clinical models of human renal cell carcinoma. Materials and Methods: Replication-deficient recombinant adenoviral vectors containing the Rous sarcoma virus promoter driving CD (Ad-RSV-CD) or TK (Ad-RSV-TK) gene expression were constructed and tested for in vitro cell-killing assays at various viral multiplicity of infection (MOI) and in vivo for growth inhibition of a human renal cell carcinoma, SK-RC-29 models. Subcutaneous tumors of SK-RC-29 were examined by electron microscopy for presence of intercellular gap junctions. Levels of expression of the gap junctional associated connexin 43 protein in SK-RC-29, 31, 38, 42, 52 human RCC cell lines were examined by western immunoblotting. Results: In vitro cell-killing assay comparing Ad-RSV-CD/5F-C and Ad-RSV- TK/ACV at a wide range of MOI (2.5 to 20) revealed superior cell-kill by Ad- RSV-CD/5-FC over Ad-RSV-TK/ACV. Consistent with these results, we observed that Ad-RSV-CD/5-FC but not Ad-RSV-TK/ACV demonstrated a significant in vivo tumor growth inhibition. These results are corroborated by the lack of gap junctions in SK-RC-29 subcutaneous tumors by the electron microscopy and the absence of connexin-43 in all five human RCC cell lines by western immunoblotting. Conclusion: We have demonstrated in this study that Ad-RSV- CD/5-FC is superior to Ad-RSVTK/ACV for the treatment of human RCC in cell culture and animal models. The results are supported by the lack of gap junctional communication between RCC cells assessed by connexin-43 expression.

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