Abstract
Survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, is widely expressed in transformed cell lines and in many different primary cancer cells, including both hematopoietic and non-hematopoietic malignancies. It is not expressed in many non-malignant adult tissues, but is essential for fetal development, as demonstrated by conventional gene-targeting experiments in mice that show embryonic lethality at day 4–6 of development. In adult cancers, including lymphoma and many epithelial carcinomas (colon, breast, gastric) the expression level of survivin, as assayed by immunohistochemical analysis and RT-PCR, correlates with overall survival.
We have designed a novel gene therapy approach that takes advantage of the high expression levels of survivin in malignant cells, in which the survivin promoter is used to drive the expression of a suicide gene to kill cancer cells by programmed cell death. Our system is based on perforin-independent granzyme B cytotoxicity and therefore does not require pro-drug activation. We designated it SAGA, for survivin and granzyme apoptosis. We used Jurkat cells as an in vitro model for T-cell leukemia, and 697Bcl2 cells as a model for pre-B Bcl2-expressing leukemia, to show this approach is more efficient in killing leukemic cells than conventional chemotherapy. Jurkat cells responded to both vincristine therapy and SAGA whereas 697Bcl2 were unaffected by vincristine, but responded to SAGA. Cell growth curves of Jurkat cells and 697Bcl2 cells are shown in Figure 1. Our approach not only inhibits cell growth, but also induces apoptosis. We detected apoptotic events by Annexin V staining and by changes in mitochondrial potential, as early as 12 hours post-treatment. Rates of early apoptotic events are shown in Table 1. In addition to these events, we also documented DNA fragmentation and caspase-3 activation in treated cells. Cytotoxicity was clearly visible by microscopic analysis 24 hours post-treatment (Figure 2). Our results strongly suggest that survivin-driven suicide gene therapy effectively enhances cell death of leukemic blasts derived from two common sub-types of ALL, one of which expresses the potent anti-apoptotic inhibitor, Bcl-2, known to be clinically more resistant to standard therapy. Experiments evaluating the in vivo effects of SAGA in mouse leukemia models are currently underway.
Cell death at 24 hours
| Early Apoptosis | Necrosis | |||||
|---|---|---|---|---|---|---|
| control | vincristine | SAGA | control | vincristine | SAGA | |
| T-ALL | 2% | 46% | 41% | 2% | 18% | 19% |
| B-ALL | 1% | 2% | 29% | 0.4% | 1% | 30% |
| Early Apoptosis | Necrosis | |||||
|---|---|---|---|---|---|---|
| control | vincristine | SAGA | control | vincristine | SAGA | |
| T-ALL | 2% | 46% | 41% | 2% | 18% | 19% |
| B-ALL | 1% | 2% | 29% | 0.4% | 1% | 30% |
