Poly-L-Ornithine-Mediated Transfection of Human Keratinocytes

Human keratinocytes are notoriously difficult to transfect. We have optimized a method for introducing plasmid DNA into keratinocytes that pairs the polycation poly-L-ornithine with a dimethylsulfoxide (DMSO) shock. The optimum poly-L-ornithine conditions for keratinocyte transfection entailed incubating the cells with 12 μg.ml poly-L-ornithine and 10 μg DNA for 6 h, followed by a 4-min 25% DMSO shock. Based on kinetic studies, 1 h is enough time to produce 10% positive cells in transient transfections, which increases up to an average of 20% after 6 h. Transfected cells survive passaging, and marker plasmids and selection can be used to yield stable transfectants at a rate twofold higher than in cells transfected with polybrene and DMSO. Transient transfection rates were significantly higher using poly-L-ornithine/DMSO than with the polybrene/DMSO or polybrete/glycerol methods previously reported. Overall, transfection mediated by poly-L-ornithine provides an efficient and inexpensive means of transiently or stably introducing DNA into keratinocytes

p300 Alters Keratinocyte Cell Growth and Differentiation through Regulation of p21Waf1/CIP1

Background: p300 functions as a transcriptional co-activator to regulate many cellular responses such as cell growth, transformation, development and differentiation. It has been shown to affect the transcriptional activity of p53 which regulates p21 Waf1/CIP1 expression, however, the role of p300 in differentiation remains unclear. Methodology and Principal Findings: Knockdown of p300 protein with short hairpin RNA (shRNA) molecules delays human neonatal foreskin keratinocyte (HFKs) differentiation. Moreover, depletion of p300 increases the proliferative capacity of HFKs, extends the life span of cells and allows differentiated HFKs to re-enter the cell cycle. Studies indicate that depletion of p300 down-regulates the acetylation and expression of p53, and chromatin immunoprecipitation (ChIP) analysis shows that induction of p21 Waf1/CIP1 in early differentiation is a result of p300 dependent activation of p53 and that depletion of p21 Waf1/CIP1 results in the delay of differentiation and a phenotype similar to p300 depletion. Conclusions: p300 has a direct role in the control of cell growth and differentiation in primary epithelial cells, and p21 Waf1/CIP1 is an important mediator of these p300 functions

Recommendations for determining HPV status in patients with oropharyngeal cancers under TNM8 guidelines : a two-tier approach

ACKNOWLEDGEMENTS The funders had no role in study design, collection, data analysis or interpretation of the data. This work received funding from the Medical Research Council (D.Mc.C. and J.J.), the Health and Social Care Research and Development Division of the Northern Ireland Public Health Agency (D.M.c.C., J.J., M.Mo.), the Wellcome Trust through the Wellcome-FDS Research Training Fellowship, the Faculty of Dental Surgery of the Royal College of Surgeons of England (A.G.S.) and GlaxoSmithKline Ltd (T.J.). The Northern Ireland OPSCC TMAs used in this research were received from the Northern Ireland Biobank which has received funds from Health and Social Care Research and Development Division of the Public Health Agency in Northern Ireland and the Friends of the Cancer Centre. The Northern Ireland Cancer Registry who receives funding from the Northern Ireland Public Health Agency carried out collection of clinical data for the Northern Ireland OPSCC patients. The Faculty of Dental Surgery of the Royal College of Surgeons of England and the Liverpool Bio-innovation Hub Biobank carried out collection of clinical data for the Liverpool OPSCC patients.Peer reviewedPublisher PD

Human papillomavirus (HPV) related Oropharynx Cancer in the United Kingdom – An evolution in the understanding of disease aetiology

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers. HPV status was determined with three validated commercial tests to provide valid data for 1,474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries. The overall proportion of HPV+ OPSCC between 2002 and 2011 was 51.8% [95% confidence interval (CI), 49.3-54.4], and this remained unchanged throughout the decade [unadjusted RR = 1.00 (95% CI, 0.99-1.02)]. However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase [age-standardized rate 2002: 2.1 (95% CI, 1.9-2.2); 2011: 4.1 (95% CI, 4.0-4.3)]. Although the number of OPSCCs diagnosed within the United Kingdom from 2002 to 2011 nearly doubled, the proportion of HPV+ cases remained static at approximately 50%. Our results argue that the rapidly increasing incidence of OPSCC in the United Kingdom cannot be solely attributable to the influence of HPV. The parallel increase in HPV+ and HPV- cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented.</p

IGF-Binding Protein 2 - Oncogene or Tumor Suppressor?

The role of insulin-like growth factor binding protein 2 (IGFBP2) in cancer is unclear. In general, IGFBP2 is considered to be oncogenic and its expression is often observed to be elevated in cancer. However, there are a number of conflicting reports in vitro and in vivo where IGFBP2 acts in a tumor suppressor manner. In this mini-review, we discuss the factors influencing the variation in IGFBP2 expression in cancer and our interpretation of these findings

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6- and E7-Expressing Keratinocytes▿

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G2/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G2 damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint

Decreased Migration of Langerhans Precursor-Like Cells in Response to Human Keratinocytes Expressing Human Papillomavirus Type 16 E6/E7 Is Related to Reduced Macrophage Inflammatory Protein-3α Production

Infection with high-risk human papillomavirus (HPV) types, particularly types 16 and 18, contributes to 90% of cervical cancer cases. HPV infects cutaneous or mucosal epithelium, tissue that is monitored for microbial infection or damage by Langerhans cells. In lesions produced by HPV type 16, there is a reduction in numbers of immune cells, especially Langerhans cells. Langerhans precursor cells selectively express CCR6, the receptor for macrophage inflammatory protein 3α (MIP-3α), and function as potent immune responders to inflamed epithelium and initiators of the innate immune response. It has been reported that E6 and E7 of high-risk HPVs interfere with immune mediators in order to suppress the recruitment of immune cells and antiviral activities of infected cells. Here we show that, following proinflammatory stimulus, HPV-16 E6 and E7 inhibit MIP-3α transcription, resulting in suppression of the migration of immature Langerhans precursor-like cells. Interestingly, the E6 and E7 proteins from the low-risk HPV types also inhibited MIP-3α transcription. These results suggest that one mechanism by which HPV-infected cells suppress the immune response may be through the inhibition of a vital alert signal, thus contributing to the persistence of HPV infection