Primary Human Foreskin Keratinocytes Research Articles

195 N/TERTs replicate primary keratinocytes in barrier formation and viral infectivity

Atopic dermatitis (AD) is a skin disease characterized by an impaired epidermal barrier and increased susceptibility to cutaneous viral infections. We have previously observed that barrier disruption (caused by our tight junction disrupting peptide [TJDP®]), enhances vaccinia virus (VV) infection of primary human foreskin keratinocytes (PHFK). To evaluate the importance of AD-relevant tight junction proteins in epidermal viral infections, we sought to identify an epidermal cell line that we could genetically manipulate. We tested N/TERT cells (immortalized PHFK) to determine whether they faithfully recapitulated primary cells in both our barrier and VV infection models. We found that N/TERTs developed a robust barrier, as measured by transepithelial electrical resistance (TEER), with peak values of 300–400 ohms/cm2 observed 4-5 days after treatment with high Ca+2 (1.8mM) containing media. After exposure to our TJDP®, N/TERTs exhibited a substantial reduction in barrier compared to media controls (e.g. TEER reduction: 46% ± 13 and 68% ± 11 at Days 2 and 3, respectively; n=3). We previously observed that PHFK are more susceptible to VV when treated with TJDP® during differentiation. With N/TERTs, we observed an increase in VV susceptibility as measured by % change in plaque number (179% ± 64 at Day 2; n=3) and % of the monolayer infected (27% ± 24 vs 42% ± 20 at Day 2 for media compared to TJDP® treated samples, n=3). These observations suggest that N/TERTs behave similarly to PHFK in both our infection and barrier experiments. Future studies will focus on determining how reduction in AD-relevant TJ barrier proteins (Claudin 1, 4, 23, occludin, and zonula occludens 1) using the CRISPR/Cas9 system impacts the susceptibility of N/TERTs to viral infections (VV and herpes simplex virus).

349 S. aureus superantigens enhance viral pathogenesis of the skin epithelium

Atopic dermatitis (AD) is characterized by skin barrier dysfunction, type 2 immunity, and an altered skin microbiota with high S. aureus abundance. Eczema vaccinatum (EV) is one of the most serious AD-associated viral infections and occurs following exposure to vaccinia virus (VV; smallpox vaccine). Previously we had observed that eczema herpeticum, another AD viral complication, was more commonly observed in S. aureus colonized patients. This led us to hypothesize that S. aureus virulence factors (e.g. superantigens [SAgs]) enhance epithelial cells susceptibility to viral infections. To test this, primary human foreskin keratinocytes (PHFK) were incubated with supernatants from multiple S. aureus strains that vary in secreted virulence factors (USA300, HG003, RN4220) and then infected with VV. Treatment of PHFK with USA300 supernatant, which contained the greatest number and quantity of SAgs, resulted in a significant increase in both plaque number and size. USA300 supernatant treatment also resulted in a significant reduction in transepithelial electrical resistance (e.g. barrier function). We have identified the S. aureus SAg, SElQ, as a virulence factor of interest as it is highly produced by USA300 and was detected on the skin of 100% of AD patients in a pilot study. Treatment of PHFK with purified SElQ decreased barrier function, enhanced viral permissiveness, and altered cellular integrity and metabolism. These changes were not observed in PHFK treated with the most homologous SAgs to SElQ, namely SElK and SElM. Our findings suggest that S. aureus SAgs, and uniquely SElQ, significantly alter PHFK and enhance their permissiveness to VV infection and spread. Based on these findings, S. aureus virulence factors are potentially key contributors to EV in AD patients, and this may extend to other viral infections afflicting this population.

Human Papillomavirus Quasivirus Production and Infection of Primary Human Keratinocytes.

This protocol describes the production of human papillomavirus (HPV)-derived quasiviruses. Quasiviruses are infectious particles that are produced in 293TT packaging cells and contain a complete viral genome. We describe methods for infection of primary human keratinocytes with HPV quasiviruses, as well as assays to measure early viral DNA replication and transcription. Published 2020. U.S. Government. Basic Protocol 1: Transfection, harvest, and isolation of HPV quasiviruses Alternate Protocol 1: Packaging HPV DNA replicated in 293TT cells Alternate Protocol 2: Production of higher-purity quasivirus using the "Ripcord" method Support Protocol 1: Production of HPV minicircles Support Protocol 2: Production of recircularized HPV genomes Support Protocol 3: Screening of fractions for viral proteins Support Protocol 4: Screening of fractions for viral DNA Support Protocol 5: Measuring viral titer Support Protocol 6: Quantitation of quasivirions Basic Protocol 2: Infection of primary human foreskin keratinocytes with quasivirus Basic Protocol 3: HPV quasivirus transcription assay Basic Protocol 4: HPV quasivirus replication assay.

Establishment of Nrf2-deficient HaCaT and immortalized primary human foreskin keratinocytes and characterization of their responses to ROS-induced cytotoxicity

Nuclear factor erythroid 2-like 2 (Nrf2) is a key transcription factor responsible for the induction of cytoprotective genes when a cell is exposed to reactive oxygen species (ROS). Insufficient ROS neutralization has been associated with undesirable changes in the skin caused by age and disease. In order to mimic the pathological conditions of these oxidative stress-induced skin disorders, we established Nrf2-deficient HaCaT and immortalized human foreskin keratinocyte (iHFK) cell lines via lentiviral transduction of Nrf2-targeting short-hairpin RNAs. Their transcriptional, as well as translational blockage of Nrf2 expression, was verified by using a proteasomal inhibitor (MG132) and well-known Nrf2 activator (α-lipoic acid (ALA)). Reduced expression of NADPH dehydrogenase quinone 1 (NQO-1) and heme oxygenase 1 (HO-1) genes, which are well-characterized downstream targets of Nrf2-mediated transactivation, was also confirmed by using ALA and another Nrf2 activator, marliolide. In general, iHFK cells displayed more enhanced cytotoxicity to menadione, a ROS-generating reference compound, than HaCaT cells. In addition, the Nrf2 deficiency highly potentiated the cytotoxic effects of menadione in both HaCaT and iHFK cells. Interestingly, pretreatment of either ALA or marliolide conferred protection against the ROS induction and the subsequent development of cytotoxicity by menadione in both HaCaT and iHFK cells regardless of the Nrf2 status. These data suggest a possibility for activation of Nrf2-independent ROS detoxification pathways by either ALA or marliolide. These newly established Nrf2-deficient HaCaT and iHFK cell lines should be useful as a highly ROS-sensitive damaged skin model for the study of age-dependent cellular changes in an in vitro setting.

Cooperative role of c-MYC and E6/E7 from two molecular variants of human papillomavirus type 16 upon proliferation and in vitro transformation of primary human keratinocytes

The roles of E6 and E7 oncoproteins of Human Papillomavirus type 16 (HPV-16) in the progression of immortalized epithelial cells to invasive tumors are not fully understood. Here, we establish a novel link between E6 and E7 of two molecular variants of HPV-16 (AA and E-350G), and c-MYC, regarding the cooperation in promoting malignant transformation of primary human foreskin keratinocytes (PHK). We aimed to study the synergistic effects of E6/E7 and c-MYC upon proliferation, and the in vitro transformation potential of PHK. We evaluated cellular proliferation through the expression of the Proliferating Cell Nuclear Antigen (PCNA) protein and colony formation abilities using soft agar and low attachment plates. We observed that E-350G-c-MYC PHKs exhibited discrete higher PCNA levels and formed significantly more colonies in both soft-agar and when growth in low-adhesion culture plates. Overall, we concluded that the E-350G variant co-transfected with c-MYC might promote malignant cellular transformation with a better efficiency than the AA-c-MYC counterpart. The enhanced oncogenic properties exhibited by the E-350G-c-MYC variant offer insights into mechanisms that may operate in human cervical neoplasia, given the higher frequency of its occurrence in the progression of high-grade precursor lesions to invasive carcinomas.

Human Papillomavirus E6 and E7 oncoproteins affect the cell microenvironment by classical secretion and extracellular vesicles delivery of inflammatory mediators

The connection between chronic inflammation and risk of cancer has been supported by several studies. The development of cancer might be a process driven by the presence of a specific combination of inflammatory mediators, including cytokines, chemokines and enzymes, in the tumor microenvironment. Virus-induced tumors, like HPV-induced Squamous Cell Carcinomas, represent a paradigmatic example of the interplay between inflammation, as integral part of the innate antiviral response, and malignant transformation. Here, the role of inflammatory microenvironment in the HPV-induced carcinogenesis is addressed, with a specific focus on the involvement of the immune molecules as well as their delivery through the microvesicle cargo possibly correlated to the different HPV genotype.The expression of the inflammatory mediators in HPV positive cells has been analyzed in primary human foreskin keratinocytes and keratinocytes transduced by E6 and E7 from mucosal HPV-16 or cutaneous HPV-38 genotypes. HPV E6 and E7 proteins can modulate the expression of immune mediators in HPV-infected cells and can affect the levels of immune molecules, mainly chemokines, in the extracellular milieu. HPV-16 E6 and E7 oncoproteins have been silenced to confirm the specificity of the modulation of the inflammatory microenvironment.Our results suggest that the expression of HPV oncoproteins allows the modification of the tumor milieu through the synthesis and release of specific pro-inflammatory cytokines and chemokines, affecting the efficacy of the immune response. The microenvironment can also be conditioned by an altered mRNA cargo delivered by extracellular vesicles, thereby efficiently affecting the surrounding cells with possible implication for tumorigenesis and tumor diagnosis.

Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction.

In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.

Cell Type- and Tissue Context-dependent Nuclear Distribution of Human Ago2

Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression.

Poly(I:C) induces controlled release of IL-36γ from keratinocytes in the absence of cell death.

The epithelium is part of an integrated immune system where cytokines, toll-like receptors and their ligands, and extracellular vesicles play a crucial role in initiating an innate immune response. IL-36γ is a pro-inflammatory member of the IL-1 family that is mainly expressed by epithelial cells, but regulation of its expression and release are only beginning to be understood. Previous studies reported that IL-36γ is abundant in recurrent respiratory papillomatosis, a rare but devastating disease caused by human papillomaviruses (HPV) types 6 and 11, in which papillomas recurrently grow in and block the airway. Despite the overexpression of IL-36γ, papilloma tissues show no evidence of inflammation, possibly due to suppression of its release by HPVs. We have used primary human foreskin keratinocytes as a model to study IL-36γ regulation in normal epithelial cells. Low doses of poly(I:C) mediate expression and release of IL-36γ without inducing the cell death reported by those using high doses. PKR, an enzyme required for inflammasome activation, does not contribute to controlled release of IL36γ. The keratinocytes secrete IL-36γ in two forms, soluble and in extracellular vesicles. We conclude that there are two separately regulated pathways for the controlled secretion of IL-36γ from keratinocytes, which could contribute to the modulation of both local and systemic immune responses to viruses and other pathogens.

ID: 109: Pro-inflammatory cytokines and chemokines in HPV-positive cancer cells

Inflammation is a critical component of tumorigenesis. Tumor microenvironment participates in the neoplastic process fostering proliferation, survival and migration. Virus-induced tumors, like HPV-induced Squamous Cell Carcinomas, represent a paradigmatic example of the interplay between inflammation, as integral part of the innate antiviral response, and malignant transformation. To study the tumorigenic role of inflammatory mediators in HPV + cells, we analyzed by real time RT-PCR the expression of selected inflammatory cytokines, chemokines and related molecules in human foreskin keratinocytes transduced by E6 and E7 from mucosalHPV-16 (K16) or cutaneousHPV-38 (K38) genotypes comparing them to primary Human Foreskin Keratinocytes. We also performed silencing experiments by using E6/E7 siRNA in HPV + SiHa carcinoma cells to verify the involvement of the viral proteins. IL-1 α and IL-1 β mRNAs are downregulated in K16 and K38 cells, whereas IL-1R1 mRNA level is comparable in all cell lines. IL-6 mRNA level is unchanged in K16 and downregulated in K38. E6/E7 silencing in SiHa confirms the effect specificity. MIP-3 α , CCL-17 and CCl-22 mRNA levels in K16 cells are also deregulated. Deregulation of MIP-3 α mRNA appears to be related to miR-21 over-expression detected in both K16 and K38. The effect of the IFN- β on the levels of these pro-inflammatory mediators will be also discussed. Experiments on paraffin embedded tissues are in progress to verify MIP-3 α deregulation in vivo. Our results suggest that HPV is able to modify the tumor microenvironment through the synthesis and release of specific pro-inflammatory cytokines and chemokines possibly to interfere with the leucocytes trafficking and/or allowing a better tumor growth and infiltration.

P63 drives invasion in keratinocytes expressing HPV16 E6/E7 genes through regulation of Src-FAK signalling.

Using microarray information from oro-pharyngeal data sets and results from primary human foreskin keratinocytes (HFK) expressing Human Papilloma Virus (HPV)-16 E6/E7 proteins, we show that p63 expression regulates signalling molecules which initiate cell migration such as Src and focal adhesion kinase (FAK) and induce invasion in 3D-organotypic rafts; a phenotype that can be reversed by depletion of p63. Knockdown of Src or FAK in the invasive cells restored focal adhesion protein paxillin at cell periphery and impaired the cell migration. In addition, specific inhibition of FAK (PF573228) or Src (dasatinib) activities mitigated invasion and attenuated the expression/activity of matrix metalloproteinase 14 (MMP14), a pivotal MMP in the MMP activation cascade. Expression of constitutively active Src in non-invasive HFK expressing E6/E7 proteins upregulated the activity of c-Jun and MMP14, and induced invasion in rafts. Depletion of Src, FAK or AKT in the invasive cells normalised the expression/activity of c-Jun and MMP14, thus implicating the Src-FAK/AKT/AP-1 signalling in MMP14-mediated extra-cellular matrix remodelling. Up-regulation of Src, AP-1, MMP14 and p63 expression was confirmed in oro-pharyngeal cancer. Since p63 transcriptionally regulated expression of many of the genes in this signalling pathway, it suggests that it has a central role in cancer progression.

Methylation-mediated repression of PRDM14 contributes to apoptosis evasion in HPV-positive cancers

Promoter methylation of the transcription factor PRDM14 (PRDI-BF1 and RIZ domain containing 14) represents a highly frequent event in human papillomavirus (HPV)-induced cervical cancers and cancer precursor lesions. Here, we aimed to assess the functional consequences of PRDM14 promoter methylation in HPV-induced carcinogenesis. PRDM14 promoter methylation, expression and consequences of ectopic PRDM14 expression were studied in HPV16-positive cervical and oral cancer cell lines (SiHa, CaSki and 93VU147T), human embryonic kidney 293 (HEK293T) cells and primary human foreskin keratinocytes (HFK). PRDM14 mRNA expression was restricted to HEK293T and HFK cells, and could be upregulated in SiHa cells upon DNA methylation inhibition. Ectopic expression of PRDM14 in SiHa, CaSki and 93VU147T cells resulted in significantly more apoptotic cells, as measured by annexin V labelling, compared to HEK293T and HFK cells. MRNA profiling of 41 apoptosis regulators identified NOXA and PUMA as candidate target genes involved in PRDM14-mediated apoptosis induction. Full-length PRDM14 transactivated both NOXA and PUMA promoters. Transactivation was abolished upon deletion of the PRDM14 DNA binding domain. This suggests that NOXA and PUMA expression is directly regulated by PRDM14, which in case of NOXA was linked to a consensus PRDM14 binding motif in the promoter region. Taken together, these results suggest that PRDM14 acts as a regulator of NOXA and PUMA-mediated apoptosis induction, thereby providing evidence for a tumour suppressive role in HPV-induced carcinogenesis. The contribution of methylation-mediated gene silencing of PRDM14 to apoptosis evasion in HPV-positive cancer cells offers novel therapeutic options for HPV-induced cancers.

Single cell in situ detection and quantification of metal oxide nanoparticles using multimodal correlative microscopy.

Assessing in situ nanoparticles (NPs) internalization at the level of a single cell is a difficult but critical task due to their potential use in nanomedicine. One of the main actual challenges is to control the number of internalized NPs per cell. To in situ detect, track, and above all quantify NPs in a single cell, we propose an approach based on a multimodal correlative microscopy (MCM), via the complementarity of three imaging techniques: fluorescence microscopy (FM), scanning electron microscopy (SEM), and ion beam analysis (IBA). This MCM was performed on single targeted individual primary human foreskin keratinocytes (PHFK) cells cultured and maintained on a specifically designed sample holder, to probe either dye-modified or bare NPs. The data obtained by both FM and IBA on dye-modified NPs were strongly correlated in terms of detection, tracking, and colocalization of fluorescence and metal detection. IBA techniques should therefore open a new field concerning specific studies on bare NPs and their toxicological impact on cells. Complementarity of SEM and IBA analyses provides surface (SEM) and in depth (IBA) information on the cell morphology as well as on the exact localization of the NPs. Finally, IBA not only provides in a single cell the in situ quantification of exogenous elements (NPs) but also that all the other endogenous elements and the subsequent variation of their homeostasis. This unique feature opens further insights in dose-dependent response analyses and adds the perspective of a better understanding of NPs behavior in biological specimens for toxicology or nanomedicine purposes.

Roles of the PDZ domain-binding motif of the human papillomavirus type 16 E6 on the immortalization and differentiation of primary human foreskin keratinocytes

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI1, MAGI2, and MAGI3, MUPP1, 14-3-3zeta, Na/H exchange regulatory factor 1, PTPN13, TIP-2/GIPC, Tip-1, and PATJ. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. However, contribution of degradation of PDZ proteins by E6 to HPV-induced oncogenesis is still controversial. In order to clarify potential roles of molecular interactions between high-risk HPV E6 and one of best characterized PDZ proteins, hDlg in HPV-induced transformation, we used a retroviral infection system to overexpress HPV16 E7 gene alone or together with either HPV16 E6 wild type or E6 mutant gene lacking the PDZ domain-binding motif and investigated the effect of mutating the PDZ domain-binding motif of E6 on the immortalization and differentiation of human foreskin keratinocytes (HFKs) by the high-risk type HPV E6 and E7. Although the PDZ domain-binding motif of E6 was found to be required for the efficient growth of HFKs, it was not necessary for the E6 and E7-induced immortalization of HFKs. Furthermore, the overexpression of E6 and E7 neither induced degradation nor altered cellular localization of hDlg in undifferentiated or differentiated HFKs. These data indicate that the PDZ domain-binding motif of E6 contributes to the efficient cellular growth through mechanisms other than degradation and changes in the subcellular localizations of hDlg.

Human papillomavirus infection is inhibited by host autophagy in primary human keratinocytes

Human papillomavirus (HPV) infection is severely limited in its natural host, primary human keratinocytes. Our data show HPV infectivity in primary keratinocytes is over 100- and 1,000-fold lower than in established keratinocyte cell lines NIKS and HaCaT, respectively. Here, we show that the basal level of autophagy in primary human foreskin keratinocytes (HFKs) is higher than in immortalized keratinocytes, and that HPV16 virions significantly induce autophagy in HFKs. Interestingly, HPV16 infectivity is dramatically enhanced by knockdown of essential autophagy genes as well as biochemical inhibition of autophagy. The increase in HPV16 infectivity by autophagy inhibition is most significant in HFKs, showing an inverse correlation with basal HPV16 infectivity in HFK, NIKS, HaCaT, and 293FT cells. Further, inhibition of autophagy delays degradation of HPV16 capsid proteins during virus trafficking, indicating that host autophagy induced by HPV16 virions inhibits infection of primary keratinocytes through rapid degradation of viral capsid proteins.

The Asian-American E6 Variant Protein of Human Papillomavirus 16 Alone Is Sufficient To Promote Immortalization, Transformation, and Migration of Primary Human Foreskin Keratinocytes

We examined how well the human papillomavirus (HPV) E6 oncogene can function in the absence of the E7 oncogene during the carcinogenic process in human keratinocytes using a common HPV variant strongly associated with cervical cancer: the Asian-American E6 variant (AAE6). This E6 variant is 20 times more frequently detected in cervical cancer than the prototype European E6 variant, as evidenced by independent epidemiological data. Using cell culture and cell-based functional assays, we assessed how this variant can perform crucial carcinogenesis steps compared to the prototype E6 variant. The ability to immortalize and transform primary human foreskin keratinocytes (PHFKs) to acquire resilient phenotypes and the ability to promote cell migration were evaluated. The immortalization capability was assayed based on population doublings, number of passages, surpassing mortality stages 1 and 2, human telomerase reverse transcriptase (hTERT) expression, and the ability to overcome G(1) arrest via p53 degradation. Transformation and migration efficiency were analyzed using a combination of functional cell-based assays. We observed that either AAE6 or prototype E6 proteins alone were sufficient to immortalize PHFKs, although AAE6 was more potent in doing so. The AAE6 variant protein alone pushed PHFKs through transformation and significantly increased their migration ability over that of the E6 prototype. Our findings are in line with epidemiological data that the AA variant of HPV16 confers an increased risk over the European prototype for cervical cancer, as evidenced by a superior immortalization, transformation, and metastatic potential.

P53 Acts as a Co-Repressor to Regulate Keratin 14 Expression during Epidermal Cell Differentiation

During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.

Oncogenic potential diverge among human papillomavirus type 16 natural variants

We compared E6/E7 protein properties of three different HPV-16 variants: AA, E-P and E-350G. Primary human foreskin keratinocytes (PHFK) were transduced with HPV-16 E6 and E7 and evaluated for proliferation and ability to grow in soft agar. E-P infected keratinocytes presented the lowest efficiency in colony formation. AA and E-350G keratinocytes attained higher capacity for in vitro transformation. We observed similar degradation of TP53 among HPV-16 variants. Furthermore, we accessed the expression profile in early (p5) and late passage (p30) transduced cells of 84 genes commonly involved in carcinogenesis. Most differences could be attributed to HPV-16 E6/E7 expression. In particular, we detected different expression of ITGA2 and CHEK2 in keratinocytes infected with AA and AA/E-350G late passage cells, respectively, and higher expression of MAP2K1 in E-350G transduced keratinocytes. Our results indicate differences among HPV-16 variants that could explain, at least in part, differences in oncogenic potential attributed to these variants.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion.

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions.

Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation.

The p63 transcription factor (TP63) is critical in development, growth and differentiation of stratifying epithelia. This is highlighted by the severity of congenital abnormalities caused by TP63 mutations in humans, the dramatic phenotypes in knockout mice and de-regulation of TP63 expression in neoplasia altering the tumour suppressive roles of the TP53 family. In order to define the normal role played by TP63 and provide the basis for better understanding how this network is perturbed in disease, we used chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq) to identify >7500 high-confidence TP63-binding regions across the entire genome, in primary human neonatal foreskin keratinocytes (HFKs). Using integrative strategies, we demonstrate that only a subset of these sites are bound by TP53 in response to DNA damage. We identify a role for TP63 in transcriptional regulation of multiple genes genetically linked to cleft palate and identify AP-2alpha (TFAP2A) as a co-regulator of a subset of these genes. We further demonstrate that AP-2gamma (TFAP2C) can bind a subset of these regions and that acute depletion of either TFAP2A or TFAP2C alone is sufficient to reduce terminal differentiation of organotypic epidermal skin equivalents, indicating overlapping physiological functions with TP63.