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Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.

The constitutive expression of human cathelicidin LL-37 antimicrobial peptide was achieved using the methylotrophic yeast, Pichia pastoris. An LL-37 cDNA clone was amplified by PCR using human fetal cDNA library as template. The 111 bp fragment encoding mature LL-37 gene was subcloned into pGAPZ-E, an episomal form of the pGAPZB vector incorporating PARS1. It was then transformed into the P. pastoris X-33 strain for intracellular expression. A small peptide with a molecular mass of about 5 kDa was detected by 17% peptide-PAGE analysis. The recombinant LL-37 peptide was purified from the gel and its amino acid sequence was determined by LC-ESI-MS/MS analysis. The initiating amino acid, methionine, was still attached to the N-terminal region of recombinant LL-37. LL-37 crude extract from P. pastoris showed an antimicrobial activity against Micrococcus luteus as the test strain. The successful expression of human LL-37 indicates that the system may be applicable to the expression of other human defensins without resorting to fusion protein constructions.

Chromosomal translocations resulting in the expression of chimaeric transcription factors are frequently observed in tumour cells, and have been suggested to be a common mechanism in human carcinogenesis. Ewing sarcoma and related peripheral primitive neuroectodermal tumours share recurrent translocations that fuse the gene EWSR1 (formerly EWS) from 22q-12 to FLI1 and genes encoding other ETS transcription factors (which bind DNA through the conserved ETS domain). It has been shown that transduction of the gene EWSR1-FLI1 (encoding EWS-FLI1 protein) can transform NIH3T3 cells, and that mutants containing a deletion in either the EWS domain or the DNA-binding domain in FLI1 lose this ability. This indicates that the EWS-FLI1 fusion protein may act as an aberrant transcription factor, but the exact mechanism of oncogenesis remains unknown. Because ETS transcription factors regulate expression of TGFBR2 (encoding the TGF-beta type II receptor, TGF-beta RII; Refs 9,14), a putative tumour suppressor gene, we hypothesized that TGFBR2 may be a target of the EWS-FLI1 fusion protein. We show here that Ewing sarcoma [corrected] (ES) cell lines with the EWSR1-FLI1 fusion have reduced TGF-beta sensitivity, and that fusion-positive ES cells and primary tumours both express low or undetectable levels of TGFBR2 mRNA and protein product. Co-transfection of FLI1 and the TGFBR2 promoter induces promoter activity, whereas EWSR1-FLI1 leads to suppression of TGFBR2 promoter activity and FLI1-induced promoter activity. Introduction of EWSR1-FLI1 into cells lacking the EWSR1-FLI1 fusion suppresses TGF-beta RII expression, whereas antisense to EWSR1-FLI1 in ES cell lines positive for this gene fusion restores TGF-beta RII expression. Furthermore, introduction of normal TGF-beta RII into ES cell lines restores TGF-beta sensitivity and blocks tumorigenicity. Our results implicate TGF-beta RII as a direct target of EWS-FLI1.

Transforming growth factor-beta (TGF-beta) is the prototypical multifunctional cytokine, participating in the regulation of vital cellular activities such as proliferation and differentiation as well as a number of basic physiological functions. The effects of TGF-beta are critically dependent on the expression and distribution of a family of TGF-beta receptors, the TGF-beta types I, II, and III. It is now known that a wide variety of human pathology can be caused by aberrant expression and function of these receptors. The coding sequence of the type II receptor (RII) appears to render it uniquely susceptible to DNA replication errors in the course of normal cell division. By virtue of its key role in the regulation of cell proliferation, TGF-beta RII should be considered as a tumor suppressor gene. High levels of mutation in the TGF-beta RII gene have been observed in a wide range of primarily epithelial malignancies, including colon and gastric cancer. It appears likely that mutation of the TGF-beta RII gene may be a very critical step in the pathway of carcinogenesis.

A 2.5-kilobase cDNA clone that encodes a 371-amino acid novel transcription factor was isolated from a human placenta cDNA library using a yeast one-hybrid system. The novel ets-related transcription factor (ERT) showed a homology with the ETS DNA-binding domain. Using constructs of the transforming growth factor-beta (TGF-beta) type II receptor (RII) promoter linked to the luciferase gene, we have demonstrated that ERT activates transcription of the TGF-beta RII gene through the 5'-TTTCCTGTTTCC-3' response element spanning nucleotides +13 to +24 and multiple additional ETS binding sites between -1816 and -82 of the TGF-beta RII promoter. A specific interaction between ERT and the ETS binding sites was also demonstrated using an electrophoretic mobility shift assay. Deletion mapping of ERT protein suggests that the transactivation domain resides in the amino terminus while the DNA-binding domain is localized to the carboxyl-terminal region. Our results suggest that ERT might be a major transcription factor involved in the transcriptional regulation of the TGF-beta RII gene.

Two angiostatic fusion proteins (hAE and hEA), differing in tandem connection manners, were constructed from human angiostatin (hAS) and endostatin (hES) proteins. These fusion proteins were then evaluated for synergistic antiangiogenic properties. The 65 kDa secreted fusion proteins, expressed in Pichia pastoris, were verified by both mass analysis and Western blotting assay. Luciferase reporter gene assay, using a VEGF promoter, revealed that the angiostatin-endostatin fusion protein (hAE), and its corresponding fusion gene delivery on human microvascular endothelial cells (HMEC-1), resulted in a more potent synergistic antiangiogenic effect than the endostatin-angiostatin fusion protein (hEA). These results suggest that the orientation of the fusion genes in hAS and hES might be an important factor in the development of therapeutic proteins.

The epithelium-specific transcription factor, ERT/ESX/ESE-1/ELF3, binds to the TGF-beta RII promoter in a sequence specific manner and regulates its expression. In this study, we investigated whether ERT could regulate endogenous TGF-beta RII expression in Hs578t breast cancer cells. Analyses of the Hs578t parental cell line revealed low RII mRNA expression and resistance to the growth inhibitory effects of TGF-beta. Infection of this cell line with a retroviral construct expressing ERT induced higher levels of endogenous RII mRNA expression and protein expression relative to cells infected with chloramphenicol acetyltransferase (CATneo) as a control. Relative to control cells, the ERTneo-expressing Hs578t cells show approximately a 50% reduction in cell growth in the presence of exogenous TGF-beta1, as well as a fourfold higher induction of activation in transient transfection assays using the 3TP-luciferase reporter construct. When transplanted into athymic mice, ERT-expressing Hs578t cells showed decreased and delayed tumorigenicity compared with control cells. This data strongly suggests that ERT plays an important role as a transcriptional activator of TGF-beta RII expression, and that deregulated ERT expression may play a critical role in rendering Hs578t human breast cancer cells insensitive to TGF-beta's growth inhibitory effects.