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Targeting of cytokine gene expression to malignant melanoma cells using tissue specific promoter sequences
1Biology of Metastasis Laboratory, Imperial Cancer Research Fund Laboratories, London, UK.
Background:
Transduction of tumor cells in vitro with cDNAs encoding various cytokines and/or immune accessory molecules has been shown to diminish or eliminate tumorigenicity when such cells are returned in vivo to syngeneic animals. One method being explored for in situ gene therapy is to use tissue-specific promoters to direct expression of the therapeutic genes to the tumour cells.
Design:
This study used the 5' flanking region of the murine tyrosinase gene to direct expression of three different cytokine genes [murine interleukin 2 (IL-2), IL-4 and macrophage colony-stimulating factor (M-CSF)] specifically to murine melanoma cells.
Results:
Expression of the IL-2 gene, from 2.5 kbp of the 5' flanking region of the murine tyrosinase gene, was detected in 11 out of 55 puromycin-resistant B16 clones isolated after transfection. The highest producing clone secreted 2000 pg/ml/10(6) cells/48 hours as determined by enzyme-linked immunosorbent assay. The IL-2 was tested for biological activity by its ability to stimulate proliferation of the IL-2 dependent CTLL cell line. No detectable level of IL-2 expression occurred in 58 clones of drug-resistant NIH 3T3 cells derived after transfection with the same construct. Similar results were obtained following transfection of these two cell lines with the tyrosinase-IL-4 minigene construct. Expression of IL-2 in the murine melanoma cells completely abrogated their tumorigenicity in syngeneic mice. However, progressively growing tumours were produced from clones in which the IL-2 gene was no longer expressed (as determined by reverse transcriptase polymerase chain reaction). Direct injection of DNA encoding cytokine genes, expressed from the tyrosinase promoter, into established B16 melanomas in syngeneic mice resulted in gene expression within the tumour mass. While no change in tumour growth was observed following such treatment, the results demonstrate that direct injection of naked DNA into a neoplasm can result in uptake and expression of cytokine genes up to 16 days post-injection.
Conclusion:
The use of tissue-specific promoters can limit expression to the required target cell, while the choice of appropriate gene should result in an alteration in tumour burden.
Insights
This study demonstrates that using the tyrosinase gene promoter to express interleukin-2 (IL-2) in melanoma cells can eliminate tumors. However, tumor recurrence occurred when IL-2 expression ceased, highlighting the need for sustained gene expression in cancer gene therapy.
Area of Science:
- Oncology
- Immunology
- Gene Therapy
Background:
- In vitro transduction of tumor cells with cytokine genes can reduce tumorigenicity in vivo.
- Tissue-specific promoters offer a strategy for targeted in situ gene therapy.
Purpose of the Study:
- To utilize the murine tyrosinase gene promoter for targeted expression of cytokine genes (IL-2, IL-4, M-CSF) in murine melanoma cells.
- To assess the therapeutic potential of cytokine gene expression in melanoma models.
Main Methods:
- Transfection of B16 melanoma cells and NIH 3T3 cells with constructs containing the tyrosinase 5' flanking region linked to cytokine genes.
- Selection of drug-resistant clones and measurement of cytokine expression using ELISA and biological assays.
- Evaluation of tumorigenicity in vivo and direct injection of DNA into established tumors.
Main Results:
- Interleukin-2 (IL-2) gene expression was achieved in B16 melanoma cells, with the highest clone secreting 2000 pg/ml/10(6) cells/48 hours.
- IL-2 expression abrogated tumorigenicity in syngeneic mice, but tumors regrew when IL-2 expression was lost.
- Direct injection of cytokine gene DNA into established melanomas led to gene expression within the tumor mass.
Conclusions:
- Tissue-specific promoters, like the tyrosinase gene promoter, enable targeted gene expression in cancer cells.
- Sustained expression of therapeutic genes is crucial for long-term tumor control in gene therapy approaches.