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Tight transcriptional regulation of transferred bacterial toxin genes represents a potential approach for gene therapy of cancer. We have previously shown that the gene for wild type diphtheria toxin A chain (DT-A) placed under transcriptional control of a tetracycline-responsive promoter cannot be silenced due to its extreme toxicity. We now have explored a tetracycline-regulated DT-A mutant involving the histidine-21 catalytic domain (H21A) which shows 120-fold reduced ADP-ribosylation activity. Cellular toxicity was determined in NIH 3T3 fibroblasts and C6 glioma cells after triple transfections with the DT-A construct, the Tet transactivator gene and a luciferase plasmid as the reporter. Marked toxicity, i.e. reduced luciferase expression by more than 98%, was observed both in the absence and in the presence of tetracycline, suggesting leakiness of the Tet system, and absence of regulation, possibly due to inhibition of DT-A synthesis by activated DT-A itself. In contrast, the lacZ gene which was driven by the same promoter could be regulated by up to 49-fold. We conclude that (1) expression but not toxicity of the DT-A mutant can be sufficiently controlled by a tetracycline-responsive promoter, and (2) tight regulation of transferred genes encoding toxins remains a challenge for gene therapy of cancer.

We studied the immunohistochemical expression of integrin alpha and beta chains in the normal and neoplastic human brain. Normal astrocytes expressed alpha 2, alpha 3, alpha 6, beta 1, and beta 4 chains in some areas facing major interstitial tissues, but they were consistently negative for the other integrins examined (alpha 4, alpha 5, alpha V, alpha L, alpha M, alpha X, beta 2, beta 3). Neoplastic astrocytes in vivo and in vitro showed increased expression of alpha 3 and beta 1, and some also of alpha 5, alpha V, beta 3, and beta 4. Neoexpression of alpha 4 and reduced levels of beta 4 were detected in glioblastoma vascular proliferations compared with normal endothelial cells. Oligodendroglioma, ependymoma, choroid plexus papilloma, pituitary adenoma, and meningioma cells showed the same integrin pattern as their normal counterparts. Adhesion assays using the astrocytoma cell lines U-138 MG and U-373 MG revealed strong attachment to collagen types I to VI and undulin, which was inhibited by antibodies to beta 1, but not by those to alpha 2, alpha 3, alpha 6, and alpha V. We conclude that astrocytomas show increased levels or neoexpression of various integrins and strong attachment to various extracellular matrix components, which appears to be almost exclusively mediated by beta 1-integrins.