BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 242, 608–612 (1998) ARTICLE NO. RC978014 Expression of Antimicrobial Peptides Has an Antitumour Effect in Human Cells David Winder,* Walter H. Gu ¨ nzburg,† , ‡ Volker Erfle,† and Brian Salmons* *Bavarian Nordic Research Institute, D-80807 Munich, Germany; †Institute of Molecular Virology, GSF-National Research Center for Environment and Health, D-85764 Oberschleissheim, Germany; and ‡Institute of Virology, University of Veterinary Sciences, A1210 Vienna, Austria Received December 13, 1997 In all of these studies, relatively high concentrations The antimicrobial peptides cecropin and melittin of purified antimicrobial peptide was administered di- are known to exhibit antitumour activity in tumour rectly, either to cells in culture or to mice. However, to derived cell lines. To achieve a similar effect in vivo be of use in therapeutic protocols, these peptides would these peptides would have to be given repeatedly to have to be repeatedly administered to maintain the maintain therapeutic levels, which may be pharmaco- high concentrations required for a beneficial effect. logically unfavourable. The expression of the genes Further, this kind of generalized delivery would result encoding such antimicrobial peptides in the desired in the amphipathic peptide reaching not only target cell type may circumvent these problems. Expression cells but also other cells. Thus, it would be advanta- constructs carrying cecropin or melittin have been geous to be able to target the delivery of the antimicro- introduced into a human bladder carcinoma derived bial peptides specifically to the site where their thera- cell line and the resultant cell clones analysed for tu- peutic activity is needed. morigenicity in nude mice. Expression of cecropin re- Transfer of genes encoding antimicrobial peptides, sulted in either a complete loss of tumorigenicity in either directly to tumour cells, or to the vicinity of the some clones or reduced tumorigenicity, as measured tumour represents one strategy to circumvent the prob- by latency of tumour formation. These results suggest that vector mediated delivery of this gene to tumour lems that may be encountered when using the active cells may prove useful for cancer gene therapy.  1998 peptide. Expression constructs in which the melittin Academic Press or cecropin genes are placed under the transcriptional control of the murine leukaemia virus (MLV) promoter are reported. Introduction and expression of these pep- tides in tumour derived human bladder carcinoma cells Peptides that show antimicrobial activity have been is associated with either reduced or no tumour growth isolated from a diverse range of organisms, including when these cells are injected into nude mice. These insects and mammals (for a review see [1]). These anti- studies suggest that such antimicrobial peptides may microbial peptides, also known as peptide antibiotics, be useful in gene therapy strategies for the treatment were initially discovered due to their role in innate of cancer. immunity and, in particular, their ability to lyse bacte- ria. However some of these peptides display a wide range of biological activities and at least three classes MATERIALS AND METHODS of antimicrobial peptides have been shown to have anti- Expression vector construction. The genes encoding prepromelit- tumour activity. One such peptide, melittin, present in tin or premelittin were amplified from the plasmid pUM13/4 [9] bee venom, has been shown to revert the transformed which carries the cDNA encoding the prepromelittin gene from Apis phenotype of H-ras transformed cells [2, 3]. Two ana- mellifera using the following primers: 1 (5-ATAGACGTCAAGGAA- logues of the cecropin antimicrobial peptide, SB-7 and GGAAGCGATCGGA-3 ) carrying an Aat II site (bold), 2 (5-TAT- Shiva-1, have been shown to preferentially lyse a num- GGATCCAACCCTGTTGCCTCTTACG-3 ) carrying a BamHI site (bold), 3 (5-TCTTACATCTATGCG-GGAATTGGAGCAGTTCTGAA- ber of lymphoma and leukaemia derived cell lines [4]. 3 ) and 4 (5-AACTGCTCCAATTCC-CGCATAGATGTAAGAAA- More recently, three cecropins have been shown to be TGT-3 ). PCR was performed using primers 1 and 2 with 1 ng cytotoxic for a number of tumour derived cell lines [5]. pUM13/4 as a template for 35 cycles (each cycle: 94°C for 1 min, 57°C At least one other class of antimicrobial peptide, the for 1 min and 72°C for 2 minutes). This PCR results in amplification of the whole prepromelittin sequence (Fig. 1A), as a 254bp DNA magainins, also exhibit antitumour cell activity [6 – 8]. 0006-291X/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved. 608