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.
OBJECTIVE
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.


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.


CONCLUSIONS
We have demonstrated in this study that Ad-RSV-CD/5-FC is superior to Ad-RSV-TK/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.
Shirakawa
T
T
Department of Urology, University of Virginia Health Sciences Center, Charlottesville, USA.
Gardner
T A
TA
Ko
S C
SC
Bander
N
N
Woo
S
S
Gotoh
A
A
Kamidono
S
S
Chung
L W
LW
Kao
C
C
D000212:Acyclovir / Q000008:administration & dosage*; D000256:Adenoviridae; D000963:Antimetabolites / Q000008:administration & dosage*; D002292:Carcinoma, Renal Cell / Q000628:therapy*; D043525:Cytosine Deaminase; D005437:Flucytosine / Q000008:administration & dosage*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D007680:Kidney Neoplasms / Q000628:therapy*; D009700:Nucleoside Deaminases / Q000008:administration & dosage*; D013937:Thymidine Kinase / Q000008:administration & dosage*; D014407:Tumor Cells, Cultured
D000963:Antimetabolites; D005437:Flucytosine; D013937:Thymidine Kinase; D009700:Nucleoside Deaminases; D043525:Cytosine Deaminase; D000212:Acyclovir