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Targeting the cytotoxicity of fusogenic membrane glycoproteins in gliomas through protease–substrate interaction

Abstract

Fusogenic membrane glycoproteins (FMG) are potent therapeutic transgenes with potential utility in the gene therapy of gliomas. FMG expression constructs caused massive syncytia formation followed by cytotoxic cell death in glioma cell lines, and antitumor activity has been shown in glioma xenografts. FMG-induced fusion in glioma cells can involve heterologous cell lines including normal astrocytes and fibroblasts, therefore making targeting important. Here we report on the use of matrix metalloproteinase (MMP) cleavable linkers to target cytotoxicity of FMGs against gliomas. Expression constructs were made expressing the hyperfusogenic version of the Gibbon Ape Leukemia Virus envelope glycoprotein (GALV) linked to a blocking ligand (the C-terminal extracellular domain of CD40 ligand) via either an MMP cleavable linker (GALV M40), a factor Xa protease cleavable linker (GALV X40), or a noncleavable linker (GALV N40). Unmodified GALV expressing constructs were used as positive controls. The glioma cell lines U87, U118, and U251 previously characterized by zymography and MMP-2 activity assay as high, medium, and low MMP expressors, respectively; normal human astrocytes and the MMP-poor cell line TE671 were transfected with the GALV, GALV N40, GALV X40, and GALV M40 constructs. In contrast to unmodified GALV constructs, transfection with GALV X40 and GALV N40 constructs blocked fusion and cytotoxic cell death. Fusion occurred, however, after transfection with constructs containing MMP cleavable linkers to an extent dependent on MMP expression in the specific cell line. Use of the broad-spectrum MMP inhibitors, 1,10-phenanthroline and N-hydroxy-piperazine-carboxamide completely abolished the ability of MMP constructs to induce fusion. In cell mixing experiments, mixing of MMP-poor cell lines transfected with GALV M40 constructs with the MMP overexpressing untransfected U87 glioma cells led to partial restoration of fusion. Use of U87 supernatant did result in a similar effect. Establishment of stable tranfectants expressing the membrane-type MMPs, MT-1 MMP and MT-2 MMP did restore fusion in the MMP-poor cell line TE671 after transfection with GALV M40, thus indicating that both membrane-type MMPs and soluble MMPs activate the MMP cleavable constructs. In addition, the GALV M40 construct retained its cytotoxic activity against U87 cells in vivo, although less effectively as compared to unmodified GALV. Our data indicate that GALV-induced cytotoxicity in glioma cell lines can be blocked by display of the CD40 ligand. Incorporation of an MMP cleavable linker can selectively restore cytotoxicity in MMP expressing glioma cell lines both in vitro and in vivo, while sparing normal human astrocytes. Given the high frequency of MMP overexpression in gliomas, this represents a promising targeting strategy.

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References

  1. Galanis E et al. Use of viral fusogenic membrane glycoproteins as novel therapeutic transgenes in gliomas. Hum Gene Ther 2001; 12: 811–821.

    Article  CAS  Google Scholar 

  2. Bateman A et al. Fusogenic membrane glycoproteins as a novel class of genes for the local and immune-mediated control of tumor growth. Cancer Res 2000; 60: 1492–1497.

    CAS  Google Scholar 

  3. Higuchi H et al. Viral fusogenic membrane glycoprotein expression causes syncytia formation with bioenergetic cell death: implications for gene therapy. Cancer Res 2000; 60: 6396–6402.

    CAS  Google Scholar 

  4. Diaz RM et al. A lentiviral vector expressing a fusogenic glycoprotein for cancer gene therapy. Gene Ther 2000; 7: 1656–1663.

    Article  CAS  Google Scholar 

  5. Dorig RE, Marcil A, Chopra A, Richardson CD . The human CD46 molecule is a receptor for measles virus (Edmonston strain). Cell 1993; 75: 295–305.

    Article  CAS  Google Scholar 

  6. Olah Z et al. The cellular receptor for Gibbon ape leukemia virus is a novel high affinity phosphate transporter. J Biol Chem 1994; 269: 25426–25431.

    CAS  PubMed  Google Scholar 

  7. Forestell SP et al. Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer. Gene Ther 1994; 4: 600–610.

    Article  Google Scholar 

  8. Morling FJ, Peng KW, Cosset F-L, Russel SJ . Masking of retroviral envelope functions by oligomerizing polypeptide adaptors. Virology 1997; 234: 51–61.

    Article  CAS  Google Scholar 

  9. Peng K-W, Vile RG, Cosset F-L, Russell SJ . Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors. Gene Ther 1999; 6: 1552–1557.

    Article  CAS  Google Scholar 

  10. Hotary K et al. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J Cell Biol 2000; 149: 1309–1323.

    Article  CAS  Google Scholar 

  11. Nakada M et al. Expression and tissue localization of membrane-type 1 2 and 3 matrix metalloproteinases in human astrocytic tumors. Am J Pathol 1999; 154: 417–428.

    Article  CAS  Google Scholar 

  12. Lampert K et al. Expression of matrix metalloproteinases and their tissue inhibitors in human brain tumors. Am J Pathol 1998; 153: 429–437.

    Article  CAS  Google Scholar 

  13. Apodaca G et al. Expression of metalloproteinases and metalloproteinase inhibitors by fetal astrocytes and glioma cells. Cancer Res 1990; 50: 2322–2329.

    CAS  Google Scholar 

  14. Yamamoto M et al. Differential expression of membrane-type matrix metalloproteinase and its correlation with gelatinase A activation in human malignant brain tumors in vivo and in vitro. Cancer Res 1996; 56: 384–392.

    CAS  Google Scholar 

  15. Ye Q-Z, Johnson LL, Yu AE, Hope D . Reconstructed 19 kDa catalytic domain of gelatinase A is an active proteinase. Biochemistry 1995; 34: 4702–4708.

    Article  CAS  Google Scholar 

  16. Will H et al. The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autocatalytic activation. J Biol Chem 1996; 271: 17119–17123.

    Article  CAS  Google Scholar 

  17. Lottenberg R, Christensen U, Jackson CM, Coleman PL . Assay of coagulation proteases using peptide chromogenic and flurogenic substrates. Methods Enzymol 1981; 80: 341–361.

    Article  CAS  Google Scholar 

  18. Uhm JH, Dooley NP, Villemure JG, Yong VW . Glioma invasion in vitro: regulation by matrix metalloprotease-2 and protein kinase C. Clin Exp Metastasis 1996; 14: 421–433.

    Article  CAS  Google Scholar 

  19. Rodriguez GC et al. Regulation of invasion of epithelial ovarian cancer by transforming growth factor-β. Gynecol Oncol 2001; 80: 245–253.

    Article  CAS  Google Scholar 

  20. Peng K-W et al. A gene delivery system activatable by disease-associated matrix metalloproteinases. Hum Gene Ther 1997; 8: 729–738.

    Article  CAS  Google Scholar 

  21. Fielding AK et al. A hyperfusogenic Gibbon ape leukemia envelope glycoprotein: targeting of a cytotoxic gene by ligand display. Hum Gene Ther 2000; 6: 817–826.

    Article  Google Scholar 

  22. Buchholz CJ et al. In vivo selection of protease cleavage sites from retrovirus display libraries. Nat Biotechnol 1998; 16: 951–954.

    Article  CAS  Google Scholar 

  23. Stetler-Stevenson WG et al. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annu Rev Cell Biol 1993; 9: 541–573.

    Article  CAS  Google Scholar 

  24. Jones L, Ghaneh P, Humphreys M, Neoptolemos JP . The matrix metalloproteinases and their inhibitors in the treatment of pancreatic cancer. Ann NY Acad Sci 1999; 880: 288–307.

    Article  CAS  Google Scholar 

  25. Moser TL et al. Secretion of extracellular matrix-degrading proteinases is increased in epithelial ovarian carcinoma. Int J Cancer 1994; 56: 552–559.

    Article  CAS  Google Scholar 

  26. Hewitt RE et al. Distribution of collagenase and tissue inhibitor of metalloproteases (TIMP) in colorectal tumors. Int J Cancer 1991; 49: 666–672.

    Article  CAS  Google Scholar 

  27. Ueno H et al. Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in human invasive breast carcinomas. Cancer Res 1997; 57: 2055–2060.

    CAS  Google Scholar 

Download references

Acknowledgements

The work was supported by CA 84388-01 (EG), the Siebens Foundation and a grant from the Fraternal Order of Eagles. We thank Dr Judith O’Fallon for her help with the statistical analysis, and Suzanne Marie Facteau for expert veterinary care and also acknowledge Ms Bonny Reinmuth and Ms Gail Prechel for their help with the preparation of the manuscript.

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Johnson, K., Peng, KW., Allen, C. et al. Targeting the cytotoxicity of fusogenic membrane glycoproteins in gliomas through protease–substrate interaction. Gene Ther 10, 725–732 (2003). https://doi.org/10.1038/sj.gt.3301951

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