Nigel A J McMillan

Publications of Nigel A J McMillan

• High prevalence of human papillomaviruses in fresh frozen breast cancer samples.

  Authors: Annika Antonsson, Terrence P Spurr, Alice C Chen, Glenn D Francis, Nigel A J McMillan, Nicholas A Saunders, Michael Law, Ian C Bennett

  Journal of medical virology. 12/2011; 83(12):2157-63.

  While the etiology of breast cancer remains enigmatic, some recent reports have examined the role of human papillomavirus (HPV) in breast carcinogenesis. The purpose of this study was to determineWhile the etiology of breast cancer remains enigmatic, some recent reports have examined the role of human papillomavirus (HPV) in breast carcinogenesis. The purpose of this study was to determine the prevalence of HPV in breast cancer tissue using PCR analysis and sequencing. Fifty-four (54) fresh frozen breast cancers samples that were removed from a cohort of breast cancer patients were analyzed. Samples were tested for HPV using comprehensive PCR primers, and in situ hybridization was performed on paraffin embedded tissue sections. Findings were correlated with clinical and pathological characteristics. The HPV DNA prevalence in the breast cancer samples was 50% (27/54) with sequence analysis indicating all cases to be positive for HPV-18 type. While HPV patients were slightly younger, no correlation was noted for menopausal status or family history. HPV positive tumors were smaller with earlier T staging and demonstrated lesser nodal involvement compared to HPV negative cancers. In situ hybridization analyses proved negative. The high proportion of HPV positive breast cancers detected in this series using fresh frozen tissues cannot be dismissed, however the role of HPV in breast carcinogenesis remains unclear and may ultimately be ascertained by monitoring future breast cancer incidence amongst women vaccinated against high risk HPV types.

• Self-catalyzed degradable cationic polymer for release of DNA.

  Authors: Nghia P Truong, Zhongfan Jia, Melinda Burgess, Liz Payne, Nigel A J McMillan, Michael J Monteiro

  Biomacromolecules. 08/2011; 12(10):3540-8.

  The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradableThe controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen/phosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.

• Self-catalyzed degradation of linear cationic poly(2-dimethylaminoethyl acrylate) in water.

  Authors: Nghia P Truong, Zhongfan Jia, Melinda Burges, Nigel A J McMillan, Michael J Monteiro

  Biomacromolecules. 05/2011; 12(5):1876-82.

  We report the synthesis of a low cytotoxic polycation that maintains its cationic strength for well over a few hours then degrades into a benign polymer with nontoxic byproducts. Well-definedWe report the synthesis of a low cytotoxic polycation that maintains its cationic strength for well over a few hours then degrades into a benign polymer with nontoxic byproducts. Well-defined poly(2-dimethylaminoethyl acrylate) (PDMAEA) of five different molecular weights prepared using reversible addition-fragmentation chain transfer (RAFT) "living" radical polymerization degrades slowly over 200 h (∼8 days). As this degradation is independent of both the polymer molecular weight and solution pH, it is consistent with a self-catalyzed hydrolysis process without the need for an internal or external degradation trigger. In addition, the polymer shows little or no cytotoxicity to HeLa cells for the molecular weights of 5600 and below, even at very high polymer concentrations (equivalent to a nitrogen/phosphorus ratio of 200). Therefore, at sufficiently low molecular weights this polymer has the essential attributes (i.e., ability to autodegradable and low toxicity) for a delivery carrier suitable for DNA or siRNA.

• Vaginal delivery of siRNA using a novel PEGylated lipoplex-entrapped alginate scaffold system.

  Authors: Sherry Y Wu, Hsin-I Chang, Melinda Burgess, Nigel A J McMillan

  Journal of controlled release : official journal of the Controlled Release Society. 02/2011; 155(3):418-26.

  Sustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administeredSustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administered intravaginally are unable to reach the vaginal epithelium under normal physiological conditions. Here we have developed a novel alginate scaffold system containing muco-inert PEGylated lipoplexes to provide a sustained vaginal presence of lipoplexes in vivo and to facilitate the delivery of siRNA/oligonucleotides into the vaginal epithelium. These PEGylated lipoplex-entrapped alginate scaffolds (PLAS) were fabricated using a freeze-drying method and the entrapment efficiency, release rate, and efficacy were characterized. We demonstrated that the PLAS system had an entrapment efficiency of ~50%, which released PEGylated lipoplexes gradually both in vitro and in vivo. While the presence of alginate diminished the cell uptake efficiency of PEGylated lipoplexes in vitro, as expected, we showed a six-fold increase their uptake into the vaginal epithelium compared to existing transfection systems following intravaginal administration in mice. A significant knockdown of Lamin A/C level was also observed in vaginal tissues using siLamin A/C-containing PLAS system in vivo. Overall, our results indicated the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium.

• Human papillomavirus in benign prostatic hyperplasia and prostatic adenocarcinoma patients.

  Authors: Alice C-H Chen, Tim Waterboer, Annie Keleher, Beth Morrison, Shalini Jindal, Denise McMillan, David Nicol, Robert A Gardiner, Nigel A J McMillan, Annika Antonsson

  Pathology oncology research : POR. 01/2011; 17(3):613-7.

  The aim of this study was to determine the prevalence of human papillomavirus (HPV) types in tissue and HPV antibodies in prostatic disease. Prostate tissue samples were collected from 51 patientsThe aim of this study was to determine the prevalence of human papillomavirus (HPV) types in tissue and HPV antibodies in prostatic disease. Prostate tissue samples were collected from 51 patients diagnosed with adenocarcinoma and 11 with benign prostatic hyperplasia (BPH). All tissue samples were confirmed by histology. Plasma samples were available for 52 prostate patients. We investigated HPV DNA prevalence by PCR, and PCR positive samples were HPV type determined by sequencing. Prevalence of antibodies against twenty-seven HPV proteins from fourteen different HPV types was assessed in the plasma samples. The HPV DNA prevalence in the tissue samples was 14% (7/51) for prostate cancer samples and 27% (3/11) for BPHs. HPV-18 was the only type detected in tissue samples (10/62). No significant difference in HPV prevalence between the prostate cancer and BPH samples was found. HPV-positive cells were identified in eight of our thirteen prostate tissue slides (3/3 BPH and 5/10 adenocarcinoma) by in situ hybridisation, and the positive cells were found in epithelial cells and peripheral blood cells. Serology data showed no significant increase in levels of antibodies against any of the HPV-18 proteins tested for in prostatic disease patients. Antibodies against HPV-1, HPV-4, HPV-6 and HPV-11 were significantly higher in the group of males with prostatic disease. Our study did not show an association between prostatic disease and either presence of HPV DNA in samples or previous exposure of high-risk HPV.

• Effective Delivery of PEGylated siRNA-Containing Lipoplexes to Extraperitoneal Tumours following Intraperitoneal Administration.

  Authors: Akul Singhania, Sherry Y Wu, Nigel A J McMillan

  Journal of drug delivery. 01/2011; 2011:192562.

  Intraperitoneal (i.p.) administration of small interfering RNA (siRNA) has, to date, shown promise in treating tumours located within the peritoneal cavity. The ability of these siRNA molecules toIntraperitoneal (i.p.) administration of small interfering RNA (siRNA) has, to date, shown promise in treating tumours located within the peritoneal cavity. The ability of these siRNA molecules to reach extraperitoneal tumours following i.p. administration is, however, yet to be investigated. Here, we examined the impact of PEGylation on the biodistribution of i.p. administered nucleic acids-containing lipoplexes. We showed that in contrast to non-PEGylated liposomes, PEGylated liposomes can deliver siRNA efficiently to extraperitoneal tumours following i.p. administration, resulting in a 45% reduction in tumour size when the oncogene-targeted siRNA was used. This difference was likely contributed by the decreased uptake of PEGylated lipoplexes in the first-pass organs, and, in particular, we observed a 10-fold decrease in the macrophage uptake of these particles compared to non-PEGylated counterparts. Overall, our results indicated the potential of using PEGylated liposomes to deliver siRNA for the treatment of i.p. localized cancer with coexisting extraperitoneal metastasis.

• Dendrosome-based delivery of siRNA against E6 and E7 oncogenes in cervical cancer.

  Authors: Tathagata Dutta, Melinda Burgess, Nigel A J McMillan, Harendra S Parekh

  Nanomedicine : nanotechnology, biology, and medicine. 06/2010; 6(3):463-70.

  Although small interfering RNA (siRNA) treatment holds great promise for the treatment of cancers, the field has been held back by the availability of suitable delivery vehicles. For cervical cancerAlthough small interfering RNA (siRNA) treatment holds great promise for the treatment of cancers, the field has been held back by the availability of suitable delivery vehicles. For cervical cancer the E6 and E7 oncogenes are ideal siRNA targets for treatment. The purpose of the present study was to explore the potential of dendrosomes for the delivery of siRNA targeting E6 and E7 proteins of cervical cancer cells in vitro. Optimization of dendrimer generation and nitrogen-to-phosphate (N/P) ratio was carried out using dendrimer-fluorescein isothiocyanate oligo complexes. The optimized N/P ratios were used in formulating complexes between dendrimers and siRNA targeting green fluorescence protein (siGFP). Although formulation 4D100 (dendrimer-siRNA complex) displayed the highest GFP knockdown, it was also found to be highly toxic to cells. In the final formulation 4D100 was encapsulated into dendrosomes so as to mask these toxic effects. The optimized dendrosomal formulation (DF), DF3 was found to possess a siGFP-entrapment efficiency of 49.76% +/- 1.62%, vesicle size of 154 +/- 1.73 nm, and zeta potential of +3.21 +/- 0.07 mV. The GFP knockdown efficiency of DF3 (dendrosome) was found to be almost identical to that of 4D100, but the former was completely nontoxic to the cells. DF3 containing siRNA against E6 and E7 was found to knock down the target genes considerably, as compared with the other formulations tested. Our results imply that dendrosomes hold potential for the delivery of siRNA and that a suitable targeting strategy could be useful for applications in vivo. FROM THE CLINICAL EDITOR: siRNA treatment holds great promise for the treatment of cancers, but overall, the availability of suitable delivery vehicles remains a major issue. The purpose of this study was to explore the potential of dendrosomes for the delivery of siRNA targeting specific proteins in cervical cancer cells in vitro. The results suggest that dendrosomes hold potential for the delivery of siRNA and a suitable targeting strategy could be useful for applications in vivo.

• Rational design of immunostimulatory siRNAs.

  Authors: Michael P Gantier, Stephen Tong, Mark A Behlke, Aaron T Irving, Martha Lappas, Ulrika W Nilsson, Eicke Latz, Nigel A J McMillan, Bryan R G Williams

  Molecular therapy : the journal of the American Society of Gene Therapy. 04/2010; 18(4):785-95.

  Short-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on theirShort-interfering RNAs (siRNAs) have engendered much enthusiasm for their ability to silence the expression of specific genes. However, it is now well established that siRNAs, depending on their sequence, can be variably sensed by the innate immune system through recruitment of toll-like receptors 7 and 8 (TLR7/8). Here, we aimed to identify sequence-based modifications allowing for the design of bifunctional siRNAs with both proinflammatory and specific silencing activities, and with potentially increased therapeutic benefits. We found that the introduction of a micro-RNA (miRNA)-like nonpairing uridine-bulge in the passenger strand robustly increased immunostimulatory activity on human immune cells. This sequence modification had no effect on the silencing efficiency of the siRNA. Increased immunostimulation with the uridine-bulge design was specific to human cells, and conserved silencing efficiency required a Dicer-substrate scaffold. The increased cytokine production with the uridine-bulge design resulted in enhanced protection against Semliki Forest virus (SFV) infection, in viral assays. Thus, we characterize a design scaffold applicable to any given siRNA sequence, that results in increased innate immune activation without affecting gene silencing. Our data suggest that this sequence modification coupled with structural modification differentially recruits human TLR8 over TLR7, and could have potential application in antiviral therapies.

• Human papillomavirus DNA detected in peripheral blood samples from healthy Australian male blood donors.

  Authors: Alice Che-Ha Chen, Annie Keleher, Mary-Anne Kedda, Amanda B Spurdle, Nigel A J McMillan, Annika Antonsson

  Journal of medical virology. 11/2009; 81(10):1792-6.

  Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In lightRecent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy individuals. Blood samples were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive samples were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs; belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.

• Both treated and untreated tumors are eliminated by short hairpin RNA-based induction of target-specific immune responses.

  Authors: Wenyi Gu, Melanie Cochrane, Graham R Leggatt, Elizabeth Payne, Allison Choyce, Fang Zhou, Robert Tindle, Nigel A J McMillan

  Proceedings of the National Academy of Sciences of the United States of America. 06/2009; 106(20):8314-9.

  RNA interference (RNAi) for cancer treatment relies on the ability to directly kill cancer cells via down-regulation of target genes, but issues of delivery and efficacy have limited clinicalRNA interference (RNAi) for cancer treatment relies on the ability to directly kill cancer cells via down-regulation of target genes, but issues of delivery and efficacy have limited clinical adoption. Furthermore, current studies using immune-deficient animal models disregard potential interactions with the adaptive immune system. It has previously been observed that certain viral antigens appear to be more rapidly presented to the immune system than normal proteins due to the production of defective ribosomal products by the virus. Given that RNAi could potentially result in the generation of truncated mRNAs, we wondered whether a similar mechanism of immune presentation of a target gene was possible. Here we show that RNAi-cleaved mRNAs can be translated into incomplete protein, and if cleavage was downstream of cytotoxic T cell epitopes, resulted in increased presentation of target protein and the generation of a tumor-protective immune response. We show that mice inoculated with tumor cells treated with such short hairpin RNAs (shRNAs) were protected from subsequent challenge with untreated tumors. However, protection was only found if shRNAs were targeted downstream of the dominant cytotoxic T cell (CTL) epitope. Our work suggests that RNAi can alter immunity to targets and shows that not all tumor cells require direct RNAi exposure for treatment to be effective in vivo, pointing the way to a new class of RNAi-based therapy.

• Dendrimer Nanocarriers as Versatile Vectors in Gene Delivery.

  Authors: Tathagata Dutta, Narendra K Jain, Nigel A J McMillan, Harendra S Parekh

  Nanomedicine : nanotechnology, biology, and medicine. 06/2009;

  The successful delivery of nucleic acids to particular target sites is the challenge that is being addressed using a variety of viral and non viral delivery systems, both of which have distinctThe successful delivery of nucleic acids to particular target sites is the challenge that is being addressed using a variety of viral and non viral delivery systems, both of which have distinct advantages and disadvantages. Non viral vectors offer the advantage of safety and flexibility over viral vectors, although lacking in efficiency. Dendrimers are novel three dimensional polymers, having the ability to interact with various forms of nucleic acids such as plasmid DNA, antisense oligonucleotides, RNA; to form complexes, which protect the nucleic acid from degradation. The interaction between the dendrimers and the nucleic acids is purely electrostatic where the cationic dendrimer condenses the anionic nucleic acids. Since cell membranes are negatively charged, the net positive charge of the dendrimer nucleic acid complex determines the transfection efficiency, although highly cationic systems are also cytotoxic. The nature of the dendrimer nucleic acid complex depends on various factors like stoichiometry, concentration of dendrimer-amines and nucleic acid-phosphates, as well as bulk solvent properties like pH, salt concentration, buffer strength, and dynamics of mixing. This article aims to review the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer based transfection reagents have become routine tools for in vitro transfection but in vivo delivery of therapeutic nucleic acids remains a challenge.

• Human papillomavirus type spectrum in normal skin of individuals with or without a history of frequent sun exposure.

  Authors: Alice Che-Ha Chen, Nigel A J McMillan, Annika Antonsson

  The Journal of general virology. 12/2008; 89(Pt 11):2891-7.

  Cutaneous human papillomavirus (HPV) has been widely detected in healthy skin. Previous studies have found that UV radiation can activate several HPV types, and a possible role for cutaneous HPV inCutaneous human papillomavirus (HPV) has been widely detected in healthy skin. Previous studies have found that UV radiation can activate several HPV types, and a possible role for cutaneous HPV in the development of non-melanoma skin cancer has been suggested. This study investigated the prevalence and type-spectrum of cutaneous HPV in relation to UV radiation by studying forehead skin swab samples from 50 healthy males frequently exposed to the sun and 50 healthy males who were not frequently exposed to the sun. A questionnaire including ethnic background of the participants, history of cancers and a self-assessment of sun-exposure was also conducted and analysed. PCR with the FAP primer pair was carried out to detect HPV DNA in samples. HPV prevalence was higher in individuals who spent more time outdoors and in individuals with a history of skin cancers (P=0.044 and P=0.04, respectively). Furthermore, individuals wearing sunglasses as a means of sun protection had a lower prevalence of HPV (P=0.018). Interestingly, HPV-76 was only detected in the group without frequent sun-exposure (P=0.001). These results suggest that increased UV radiation exposure may be a factor leading to a difference in prevalence of cutaneous HPV types.

• Molecular basis of pathogenesis, prognosis and therapy in chronic lymphocytic leukaemia.

  Authors: Jiezhong Chen, Nigel A J McMillan

  Cancer biology & therapy. 03/2008; 7(2):174-9.

  B-cell chronic lymphocytic leukemia (CLL) is caused by the abnormal accumulation of non-functional B-cells in peripheral blood and bone marrow. However, the precise aetiology and mechanism of theB-cell chronic lymphocytic leukemia (CLL) is caused by the abnormal accumulation of non-functional B-cells in peripheral blood and bone marrow. However, the precise aetiology and mechanism of the disease are unclear. Recently, progress has been made in the identification of both the genetic deficiencies and environmental factors that may underlie CLL. This has provided some clues to the nature of the disease, but no definitive cures. Although treatment has increased remission time, at present the disease is not curable by conventional therapy. Further studies of the pathogenesis of CLL are needed, as are the development of suitable cell lines and animal models in which to study it. This review summarises the most recent progress in CLL with emphasis on molecular events and possible implications in therapy.

• Multiple signal pathways in the leukemogenesis and therapeutic implications.

  Authors: Jiezhong Chen, Nigel A J McMillan

  Leukemia research. 01/2008; 31(12):1759-60.

• The development and future of oligonucleotide-based therapies for cervical cancer.

  Authors: Wenyi Gu, Lisa N Putral, Aaron Irving, Nigel A J McMillan

  Current opinion in molecular therapeutics. 05/2007; 9(2):126-31.

  Cervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-basedCervical cancer is an attractive model in which to test gene-specific therapies, because elimination of the HPV oncogenes E6 and E7 may result in cancer cell senescence. Oligonucleotide-based therapies tested over the years include antisense oligonucleotides, ribozymes and, more recently, small interfering RNA (siRNA)-based treatments. The development and use of these technologies are reviewed. siRNA-based therapies have been touted as potential treatments for cancers, genetic disorders and viral infections and have a number of advantages over antisense and ribozyme technologies. As with the older technologies, in vitro testing of siRNAs against cervical cancer has shown promising results, however, the issues that held up the clinical development of ribozymes and antisense are currently also challenging the siRNA field; these are target selection, specificity and delivery. If these issues can be overcome, a range of new and potent therapies for cervical cancer could become available.

• Papillomavirus in healthy skin of Australian animals.

  Authors: Annika Antonsson, Nigel A J McMillan

  The Journal of general virology. 12/2006; 87(Pt 11):3195-200.

  Papillomaviruses are a group of ubiquitous viruses that are often found in normal skin of humans, as well as a range of different vertebrates. In this study, swab samples collected from the healthyPapillomaviruses are a group of ubiquitous viruses that are often found in normal skin of humans, as well as a range of different vertebrates. In this study, swab samples collected from the healthy skin of 225 Australian animals from 54 species were analysed for the presence of papillomavirus DNA with the general skin papillomavirus primer pair FAP59/FAP64. A total of five putative and potential new animal papillomavirus types were identified from three different animal species. The papillomaviruses were detected in one monotreme and two marsupial species: three from koalas, and one each from an Eastern grey kangaroo and an echidna. The papillomavirus prevalence in the three species was 14 % (10/72) in koalas, 20 % (1/5) in echidnas and 4 % (1/23) in Eastern grey kangaroos. Phylogenetic analysis was performed on the putative koala papillomavirus type that could be cloned and it appears in the phylogenetic tree as a novel putative papillomavirus genus. The data extend the range of species infected by papillomaviruses to the most primitive mammals: the monotremes and the marsupials.

• Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding.

  Authors: Thomas Fothergill, Nigel A J McMillan

  Virology. 10/2006; 352(2):319-28.

  We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so weWe have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 31, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3beta. We also present evidence that PV signaling is activated via the alpha6beta4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection.

• The human papillomavirus type 16 E7 protein binds human interferon regulatory factor-9 via a novel PEST domain required for transformation.

  Authors: Annika Antonsson, Elizabeth Payne, Kylie Hengst, Nigel A J McMillan

  Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research. 08/2006; 26(7):455-61.

  It is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction ofIt is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction of the HPV-16 E7 protein with IFN regulatory factor-9 (IRF-9). Here, we investigated the details of the interaction using HPV-16 E7 peptide mapping to show that IRF-9 binds HPV-16 E7 in a domain encompassing amino acids 25-36. A closer examination of this region indicates this is a novel proline, glutamate, serine, and threonine-rich (PEST) domain, with a PEST score of 8.74. We have also mapped the region of interaction within IRF-9 and found that amino acids 354-393 play an important role in binding to HPV-16 E7. This region of IRF-9 encompasses the IRF association domain (IAD), a region important for protein-protein interaction central to IRF function. Finally, we used alanine-scanning mutagenesis to determine if E7-IRF-9 interaction was important for E7-mediated cellular transformation and found that the HPV-16 E7 mutants Y25A, E26A, S31A, S32A, and E35A, but not L28A and N29A, caused loss of transformation ability. Preliminary data suggest loss of IRF-9 interaction with E7 mutants correlated with transformation. Our work suggests E7-IRF-9 interaction is important for the transforming ability of HPV-16 E7 and that HPV-16 E7 may interact with other IRF proteins that have IAD domains.

• RNA interference against human papillomavirus oncogenes in cervical cancer cells results in increased sensitivity to cisplatin.

  Authors: Lisa N Putral, Megan J Bywater, Wenyi Gu, Nicholas A Saunders, Brian G. Gabrielli, Graham R Leggatt, Nigel A J McMillan

  Molecular pharmacology. 12/2005; 68(5):1311-9.

  Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV)Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 microM, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 microM. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with individual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

• Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription.

  Authors: Liliana Endo-Munoz, Tammra Warby, David Harrich, Nigel A J McMillan

  Virology journal. 02/2005; 2:17.

  BACKGROUND: The interferon (IFN)-induced, dsRNA-dependent serine/threonine protein kinase, PKR, plays a key regulatory role in the IFN-mediated anti-viral response by blocking translation in theBACKGROUND: The interferon (IFN)-induced, dsRNA-dependent serine/threonine protein kinase, PKR, plays a key regulatory role in the IFN-mediated anti-viral response by blocking translation in the infected cell by phosphorylating the alpha subunit of elongation factor 2 (eIF2). The human immunodeficiency virus type 1 (HIV-1) evades the anti-viral IFN response through the binding of one of its major transcriptional regulatory proteins, Tat, to PKR. HIV-1 Tat acts as a substrate homologue for the enzyme, competing with eIF2alpha, and inhibiting the translational block. It has been shown that during the interaction with PKR, Tat becomes phosphorylated at three residues: serine 62, threonine 64 and serine 68. We have investigated the effect of this phosphorylation on the function of Tat in viral transcription. HIV-1 Tat activates transcription elongation by first binding to TAR RNA, a stem-loop structure found at the 5' end of all viral transcripts. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. RESULTS: We have investigated the effect of phosphorylation on Tat-mediated transactivation. Our results showed faster, greater and stronger binding of Tat to TAR RNA after phosphorylation by PKR. In vitro phosphorylation experiments with a series of bacterial expression constructs carrying the wild-type tat gene or mutants of the gene with alanine substitutions at one, two, or all three of the serine/threonine PKR phosphorylation sites, showed that these were subject to different levels of phosphorylation by PKR and displayed distinct kinetic behaviour. These results also suggested a cooperative role for the phosphorylation of S68 in conjunction with S62 and T64. We examined the effect of phosphorylation on Tat-mediated transactivation of the HIV-1 LTR in vivo with a series of analogous mammalian expression constructs. Co-transfection experiments showed a gradual reduction in transactivation as the number of mutated phosphorylation sites increased, and a 4-fold decrease in LTR transactivation with the Tat triple mutant that could not be phosphorylated by PKR. Furthermore, the transfection data also suggested that the presence of S68 is necessary for optimal Tat-mediated transactivation. CONCLUSION: These results support the hypothesis that phosphorylation of Tat may be important for its function in HIV-1 LTR transactivation.