Marcella Flinterman

Delivery of Therapeutic Proteins as Secretable TAT Fusion Products

The trans-acting activator of transcription (TAT) protein transduction domain (PTD) mediates the transduction of peptides and proteins into target cells. The TAT-PTD has an important potential as a tool for the delivery of therapeutic agents. The production of TAT fusion proteins in bacteria, however, is problematic because of protein insolubility and the absence of eukaryotic post-translational modification. An attractive alternative, both for in vitro protein production and for in vivo applications, is the use of higher eukaryotic cells for secretion of TAT fusion proteins. However, the ubiquitous expression of furin endoprotease (PACE or SPC1) in the Golgi/endoplasmic reticulum, and the presence of furin recognition sequences within TAT-PTD, results in the cleavage and loss of the TAT-PTD domain during its secretory transition through the endoplasmic reticulum and Golgi. In this study, we show the development of a synthetic TATkappa-PTD in which mutation of the furin recognition sequences, but retention of protein transduction activity, allows secretion of recombinant proteins, followed by successful uptake of the modified protein, by the target cells. This system was used to successfully secrete marker protein, green fluorescent protein (GFP), and apoptin, a protein with tumor-specific cytotoxicity. Detection of GFP, phosphorylation, and induction of cell death by TATkappa-GFP-apoptin indicated that the secreted proteins were functional in target cells. This novel strategy therefore has important potential for the efficient delivery of therapeutic proteins.

E1A-mediated suppression of EGFR expression and induction of apoptosis in head and neck squamous carcinoma cell lines

Previous studies have shown early region 1A (E1A) gene to inhibit the proliferation of tumour cells with wild-type, but not mutant, p53. E1A has also been shown to downregulate c-erb-B-2/neu expression, resulting in inhibition of growth in c-erb-B-2/neu overexpressing tumour cells. In this study, we have investigated the effect of E1A expression on four head and neck squamous cell carcinoma (HNSCC) cell lines that do not overexpress c-erb-B-2/neu. Cell cycle and Western blot analysis show E1A-mediated induction of apoptosis in all cell lines examined. This induction of apoptosis was independent of the p53 status as it occurred in the cell lines with wild-type, mutated or deleted p53. However, there was no evidence of E1A-induced apoptosis in a p53+ve normal human fibroblast cell line, 1BR3. Analysis of apoptosis in the SCC cell lines demonstrated E1A-mediated downregulation of EGFR, which was overexpressed in each of these cell lines. Overexpression of an exogenously introduced EGFR, under the control of an E1A-insensitive heterologous promoter, blocked E1A induction of apoptosis in these cells. Therefore, E1A-mediated downregulation of EGFR expression appears to be the cause, rather than a consequence of E1A-induced apoptosis in these SCC cell lines. Previous studies have shown downregulation of EGFR expression by PML. Interestingly, E1A expression in the HNSCC cells altered the pattern of PML distribution and induced the level of PML protein, thus suggesting that E1A-mediated downregulation of EGFR may occur via direct or indirect interactions with PML. These findings demonstrate a novel pathway by which E1A can induce apoptosis and identify EGFR as a potential target for the development of therapeutic strategies against epithelial malignancies, the majority of which have abnormal EGFR expression.

p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing.

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells, remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis.

Specific isoforms of p73 control the induction of cell death induced by the viral proteins, E1A or apoptin

A member of the p53 family, p73, has several isoforms and differentially regulates transcription of genes involved in the control of the cell cycle and apoptosis. We have previously shown efficient and p53-independent, tumour-specific cell death induced by the viral proteins E1A and Apoptin. Here, we demonstrate that the induction of apoptosis by these viral proteins involves activation of TAp73. Both E1A and Apoptin induced expression of endogenous TAp73 and the p53/p73 BH3-only pro-apoptotic target, PUMA, independently of the p53 function. Furthermore, exogenous expression of TAp73 isoforms, particularly TAp73β, sensitized cells to killing by both E1A and Apoptin, while expression of ΔNp73α blocked this activity. Besides, knockout of the p73 regulator, c-Abl, attenuated E1A-induced apoptosis. In accordance with the role of p73 in apoptosis induced by these viral proteins, overexpression of TAp73β strongly induced apoptosis in p53-deficient cancer cells in vitro and in HNSCC xenografts. Using a doxycycline-inducible system, we provide evidence for target selectivity and significant differences in protein stability for specific p73 isoforms, suggesting a diverse and pivotal role for p73 in response to various genotoxic agents. Collectively, our data show that in the absence of the p53 function, viral proteins E1A and Apoptin utilize the p73 pathway to induce efficient tumour cell death.

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ∆Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.

Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

Family at last: highlights of the first international meeting on proteins killing tumour cells

Recently, a select number of viral and cellular proteins sharing a unique property have been identified, namely apoptin, HAMLET, TRAIL, MDA7, E4ORF4, NS1 and Brevinin-2R, where all have the remarkable ability to selectively kill transformed and/or tumour cells, while causing no or only minor cytotoxicity in normal and non-transformed cells. Research worldwide is now starting to unravel the molecular signalling pathways by which each of these proteins exerts its tumour-selective function, and the first (pre)clinical studies are very promising. The workshop ‘Proteins Killing Tumour Cells’ brought together, for the first time, researchers working on this novel class of intriguing proteins. Participants from 14 different countries shared their views on (possible common) mechanisms behind tumour-specific apoptosis induction and strategies to implement this knowledge in the clinic.

PML involvement in the p73-mediated E1A-induced suppression of EGFR and induction of apoptosis in head and neck cancers

Epidermal growth factor receptor (EGFR) tyrosine kinase is commonly overexpressed in human cancers; however, the cellular mechanisms regulating EGFR expression remain unclear. p53, p63 and p73 are transcription factors regulating many cellular targets involved in controlling the cell cycle and apoptosis. p53 activates EGFR expression, whereas TAp63 represses EGFR transcription. The involvement of p73 in the regulation of EGFR has not been reported. Here, a strong correlation between EGFR overexpression and increased levels of the oncogenic ΔNp73 isoform in head and neck squamous cell carcinoma (HNSCC) cell lines was observed. Ectopic expression of TAp73, particularly TAp73β, resulted in suppression of the EGFR promoter, significant downregulation of EGFR protein and efficient induction of cell death in all six EGFR-overexpressing HNSCC cell lines. EGFR overexpression from a heterologous LTR promoter protected lung cancer cells from TAp73β-induced EGFR suppression and apoptosis. Expression of TAp73β efficiently induced promyelocytic leukaemia (PML) protein expression and PML knockdown by shRNA attenuated the downregulation of EGFR and induction of apoptosis by p73 in HNSCC cells. Furthermore, PML was found to be important for E1A-induced suppression of EGFR and subsequent killing of HNSCC cells. Our data therefore suggest a novel pathway involving PML and p73 in the regulation of EGFR expression.

Novel cationic polyene glycol phospholipids as DNA transfer reagents—Lack of a structure-activity relationship due to uncontrolled self-assembling processes

Cationic glycol phospholipids were synthesized introducing chromophoric, rigid polyenoic C20:5 and C30:9 chains next to saturated flexible alkyl chains of variable lengths C6-20:0. Surface properties and liposome formation of the amphiphilic compounds were determined, the properties of liposome/DNA complexes (lipoplexes) were established using three formulations (no co-lipid, DOPE as a co-lipid, or cholesterol as a co-lipid), and the microstructure of the best transfecting compounds inspected using small angle X-ray diffraction to explore details of the partially ordered structures of the systems that constitute the series. Transfection and cytotoxicity of the lipoplexes were evaluated by DNA delivery to Chinese hamster ovary (CHO-K1) cells using the cationic glycerol phospholipid 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EPC) as a reference compound. The uncontrollable self-association of the molecules in water resulted in aggregates and liposomes of quite different sizes without a structure-property relationship. Likewise, adding DNA to the liposomes gave rise to unpredictable sized lipoplexes, which, again, transfected without a structure-activity relationship. Nevertheless, one compound among the novel lipids (C30:9 chain paired with a C20:0 chain) exhibited comparable transfection efficiency and toxicity to the control cationic lipid EPC. Thus, the presence of a rigid polyene chain in this best performing achiral glycol lipid did not have an influence on transfection compared with the chiral glycerolipid reference ethyl phosphocholine EPC with two flexible saturated C14 chains.