Yonsei Med J.  2008 Oct;49(5):811-818. 10.3349/ymj.2008.49.5.811.

Imaging of Viral Thymidine Kinase Gene Expression by Replicating Oncolytic Adenovirus and Prediction of Therapeutic Efficacy

• Kim EJ
• Yoo JY
• Choi YH
• Ahn KJ
• Lee JD
• Yun CO
• Yun M

• KMID: 2158200
• DOI: http://doi.org/10.3349/ymj.2008.49.5.811

Abstract

PURPOSE
We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system. MATERIALS AND METHODS: Replication-competent recombinant adenoviral vector (Ad-deltaE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-deltaE1A) was generated as a control. Both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment. RESULTS: On gamma counter-analyses, counts/minute (cpm)/microgram of protein showed MOIs dependency with deltaE1B19/55-TK infection. On MTT assay, Ad-deltaE1B19/55-TK led to more efficient cell killing than Ad-deltaE1A-TK. On plate imaging by gamma camera, both Ad-deltaE1B19/55-TK and Ad-deltaE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of deltaE1B19/55-TK infection was remarkably reduced compared to that of Ad-deltaE1A-TK infection. CONCLUSION: Replicating Ad-deltaE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-deltaE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-deltaE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.

Keyword

Oncolysis; adenovirus; thymidine kinase; gene therapy; radiotracer

MeSH Terms

Adenoviridae/*genetics/physiology
Cell Line, Transformed
Cell Line, Tumor
Ganciclovir/pharmacology
Gene Expression
Gene Therapy/methods
Genetic Vectors
Humans
*Oncolytic Virotherapy
Oncolytic Viruses/*genetics/physiology
Simplexvirus/genetics
Tetrazolium Salts/analysis
Thiazoles/analysis
Thymidine Kinase/*genetics/metabolism
Transgenes
Viral Proteins/*genetics/metabolism
Virus Replication

Figure

• 

  Fig. 1 Characterization of adenoviruses expressing HSVtk gene. (A) Specific primer sets corresponding to E1 and HSVtk gene are shown below the diagram of E1 and E3 region of adenovirus genome, and each expected size of PCR products is 479 bp and 700 bp, respectively. (B) PCR product analysis of Ad-ΔE1A, Ad-ΔE1A-TK, Ad-ΔE1B19/55, and Ad-ΔE1B19/55-TK. The presence of each PCR product verified the expression of the E1A gene and the insertion of HSVtk gene.

• 

  Fig. 2 Measurement of TK expression by I-131 FIAU and gamma counter. The YCC-2 cells were infected with a range of titers of Ad-ΔE1B19/55-TK (■) or Ad-ΔE1A-TK (▲) for 24 to 72 hours and were incubated with I-131 FIAU. On gamma counter analyses of lysed cells, cpm/µg of protein showed MOIs dependency with a linear increase from 1 to 40 MOI at all time points with ΔE1B19/55-TK infection. With Ad-ΔE1A-TK infection, cpm /µg of protein were unchanged over MOIs after 24 hours of infection and slightly increased after 48 and 72 hours of infection with 40 MOI. MOI, multiplicity of infection.

• 

  Fig. 3 MTT assay after Ad-ΔE1B19/55-TK (■) or Ad-ΔE1A-TK (▲) infection. The cytotoxicity was determined after infection with a range of titers of Ad-ΔE1B19/55-TK or Ad-ΔE1A-TK for 24 to 72 hours. Ad-ΔE1B19/55-TK led to more efficient cell killing than Ad-ΔE1A-TK after 48 and 72 hours of infection and the amount of viruses needed was lower with Ad-ΔE1B19/55-TK for the same cytotoxicity. MOI, multiplicity of infection.

• 

  Fig. 4 (A) Gamma ray imaging using I-131 FIAU after Ad-ΔE1B19/55-TK or Ad-ΔE1A-TK infection. The cells were infected with Ad-ΔE1B19/55-TK or Ad-ΔE1A-TK at MOIs of 1, 10, and 40 for 48 hours. Both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern. (B) The differences in uptakes of I-131 FIAU between the two viral infections were larger with higher viral titers. MOI, multiplicity of infection.

• 

  Fig. 5 Combination treatment with Ad-ΔE1B19/55-TK (■) or Ad-ΔE1A-TK (▲) and GCV. GCV was added at final concentration of 100 µg/mL at 48 hours postinfection. After additional 3 days of incubation, MTT assay was performed. Cell viability of ΔE1B19/55-TK infection was remarkably reduced compared to that of Ad-ΔE1A-TK infection. MOI, multiplicity of infection; GCV, ganciclovir.

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