J Korean Med Sci.  2004 Feb;19(1):107-112. 10.3346/jkms.2004.19.1.107.

Bystander-Mediated Regression of Murine Neuroblastoma via Retroviral Transfer of the HSV-TK Gene

• Cho HS
• Lee HR
• Kim MK

• KMID: 1785703
• DOI: http://doi.org/10.3346/jkms.2004.19.1.107

Abstract

Selective introduction of genes conferring chemosensitivity into proliferating tumor cells may be used to treat cancer. We investigated the bystander effect of retrovirusmediated gene transfer of herpes simplex virus thymidine kinase (HSV-TK) gene to murine neuroblastoma cell line (neuro-2a) in vitro and in vivo, and we examined whether the mechanism of bystander effect in neuroblastoma would also depend on connexin-dependent gap junction and/or immune response. A strong bystander effect was observed in vitro, whereby nontransduced tumor cells in proximity to transduced cells acquired susceptibility to ganciclovir (GCV) killing. Implanted mixtures of wildtype cells and HSV-TK transduced cells showed a potent bystander effect upon administration of GCV in A/J mice. HSV-TK/GCV system in murine neuroblastoma induced systemic immunity. Immunohistochemical staining showed many CD4+ and CD8+ cell infiltration but did not show anti-connexin 43+ cells. In conclusion, a strong bystander effect was observed in vitro and in vivo. The bystander effect in murine neuroblastoma might be dependent on immune response and/or on other mechanism such as protein phosphorylation or transfer of apoptotic vesicle, rather than connexin-dependent gap junction.

Keyword

Bystander Effect; Neuroblastoma; Gene Therapy

MeSH Terms

Animals
Apoptosis
Bystander Effect
CD4-Positive T-Lymphocytes/metabolism
CD8-Positive T-Lymphocytes/metabolism
Cell Line, Tumor
Connexin 43/biosynthesis
Gap Junctions
Gene Therapy/*methods
*Gene Transfer Techniques
Human
Immunohistochemistry
Mice
Neoplasm Transplantation
Neuroblastoma/*therapy
Phosphorylation
Retroviridae/genetics
Simplexvirus/*enzymology
Support, Non-U.S. Gov't
Thymidine Kinase/*genetics
Time Factors

Figure

• 

  Fig. 1 In vitro bystander assay. Wild type neuro-2a cells and neuro-2a/TK cells were mixed at varying ratios, plated in 6 well culture plates (106/cell), and incubated at 37℃ overnight. Cells were exposed to GCV (10 µg/mL). On day 1, day 2, day 3 and day 4, cells were stained with trypan blue and surviving cells were enumerated.

• 

  Fig. 2 In vivo bystander effect. 2×106 cells were injected s.c. into the flank as follows: 100% HSV-TK-transduced (Group 1) and 100% untransduced cells (Group 2) and mixtures of transduced and untransduced cells at ratios of 1:1 (Group 3), 1:10 (Group 4) and 1:20 (Group 5). When the tumors reached the diameter of 5-8 mm, the mice received intraperitoneally 50 mg/kg GCV twice a day for 7 days. Group 2 versus Group 1: significant, p<0.01; Group 2 versus group 3: significant, p<0.01; Group 2 versus Group 4: significant, p<0.01; Group 2 versus Group 5: significant, p<0.05 (log rank test).

• 

  Fig. 3 Induction of immune response in HSV-TK gene-transduced neuro-2a cells. 2×106 HSV-TK cells (Group 1) and 2×106 neuro-2a cells (Group 2) injected s.c. into the flank of the mice, respectively (N=6/group). One week after tumor cell challenge, the mice received intraperitoneally 50 mg/kg GCV twice a day for 7 days and then the animals were challenged by s.c. implantation of approximately 2×106 unmodified neuro-2a cells contralaterally to the original injection sites 14 days after the last GCV treatment. Log rank test showed group survived significantly compared with Group 2 (p<0.01).

• 

  Fig. 4 Expression of CD 4, CD 8 and connexin-43 antigen. Paraffin-embedded sections were reacted with goat polyclonal antibodies to CD4 (A), rabbit polyclonal antibodies to CD8 (B) and not reacted with mouse anti-connexin 43 antibody (C). (A, B) Cell membrane staining of subcutaneous tissue of the tumor-regressed site. Magnification was at ×1,000. (C) Solid area of neuroblastoma before GCV. Magnification was at ×400.

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