In solid tumors deprived of glucose and oxygen, highly expressed proteins are glucose regulated proteins (GRPs). In particular, GRP78 is elevated about 10 fold, by transcriptional regulation. Promoters of rat and human grp78 have been isolated and shown to respond to glucose or oxygen starvation opening new approaches to targeted gene therapy of tumors. Vectors can be constructed in which the grp78 promoter drives expression of gene products able to induce local inflammation and cell death. The proposal has three goals, the first is to examine whether the grp78 promoter, serving as an internal promoter in a retroviral vector, can drive high level expression of a reporter gene in tumor cells. Retroviral vectors using either grp78 or SV40 as promoters, for the neo gene, will be transduced into B/C10ME cells and tranfectants selected by colchicine. Expression of neo under glucose starvation will be examined in vitro. Transduced cells injected into mice will be assayed for reporter gene expression and will show whether in growing tumors under stress, the grp78 promoter enhances expression of neo under its control. The grp78 promoter will be compared to the SV40 promoter, then modified to lower basal level and to optimize stress inducible transcription. These promoters will be tested in cultured cells, then assayed in vivo. The second goal is to generate vectors with the grp78 promoter to drive expression of cytokines IL-2 and GM-CSF. Cytokine secretion will be assayed under normal or stress induced conditions in vitro and in vivo. Transduced tumor cell lines will be injected into mice and tumorigenicity compared to that of parental, non-transduced cells or cells secreting cytokines constitutively. Acute rejection by NK cells and delayed rejection by CTL will be assayed. In alternative approaches C2 myogenic cells, transfected with vectors coding for cytokines under control of the grp78 promoter will be tested for ability to induce tumor regression. Similar attempts will be made by infection of tumors with packaged virus containing these plasmids. The third goal is to examine effects of GRP78 levels on tumor growth prompted by experiments showing that induction of GRP78 induces resistance to CTL cytotoxicity. Suppression of GRP78 induction by antisense vectors in B/C10ME eliminates resistance. Therefore the question arises does induction of stress protein in general enhance in vivo tumor growth? Several tumors will be assayed for sensitivity to CTL and TNF after stress induction. If resistance is induced they will be transfected with grp78 antisense constructs. Failure to induce GRP78 should lead to decreased tumor growth in vivo.
Showing the most recent 10 out of 70 publications
