In vitro three-dimensional modelling of human ovarian surface epithelial cells

Objectives: Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three-dimensional (3D) cultures. Materials and methods: OSE cells were cultured as multicellular spheroids (MCS) (i) in a rotary cell culture system (RCCS) and (ii) on polyHEMA-coated plastics. The MCSs were examined by electron microscopy and compared to OSE from primary tissues and cells grown in 2D. Annexin V FACS analysis was used to evaluate apoptosis and expression of extracellular matrix (ECM) proteins was analysed by immunohistochemical staining. Results: On polyHEMA-coated plates, OSE spheroids had defined internal architecture. RCCS MCSs had disorganized structure and higher proportion of apoptotic cells than polyHEMA MCSs and the same cells grown in 2D culture. In 2D, widespread expression of AE1/AE3, laminin and vimentin were undetectable by immunohistochemistry, whereas strong expression of these proteins was observed in the same cells grown in 3D culture and in OSE on primary tissues. Conclusions: Physiological and biological features of OSE cells grown in 3D culture more closely resemble characteristics of OSE cells in vivo than when grown by classical 2D approaches. It is likely that establishing in vitro 3D OSE models will lead to greater understanding of the mechanisms of neoplastic transformation in epithelial ovarian cancers. © 2009 Blackwell Publishing Ltd

3D in vitro model of a functional epidermal permeability barrier from human embryonic stem cells and induced pluripotent stem cells

Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generatio

Human ovarian surface epithelial cells immortalized with hTERT maintain functional pRb and p53 expression

Objective: Cell immortalization is considered to be a prerequisite status for carcinogenesis. Normal human ovarian surface epithelial (OSE) cells, which are thought to be the origin of most of human ovarian carcinomas, have a very limited lifespan in culture. Establishment of immortalized OSE cell lines has, in the past, required inactivation of pRb and p53 functions. However, this often leads to increased chromosome instability during prolonged culture. Materials and Methods: In this study, we have used a retroviral infection method to overexpress human telomerase reverse transcriptase (hTERT) gene, in primary normal OSE cells, under optimized culture conditions. Results: In vitro and in vivo analysis of hTERT-immortalized cell lines confirmed their normal epithelial characteristics. Gene expression profiles and functional analysis of p16(INK4A), p15(INK4B), pRb and p53 confirmed the presence of their intact functions. Our study suggests that inactivation of pRb and p53 is not necessary for OSE immortalization. Furthermore, down-regulation of p15(INK4B) in the immortalized cells may indicate a functional role for this protein in them. Conclusion: These immortal OSE cell lines are likely to be an important tool for studying human OSE biology and carcinogenesis

RNAi depletion of PRSS21 induces apoptosis in cutaneous squamous cell carcinoma keratinocytes

The mechanisms underlying UVB-induced clearance of psoriasis are incompletely understood. We compared the cellular and molecular effects of a clinically effective wavelength of UVB (311nm) with a clinically ineffective wavelength (290nm) in vivo and in vitro to distinguish bystander effects and utilised a systems biology approach to understand functional significance. 84 adult psoriatic patients were recruited. Biopsies were taken from lesional (plaque) skin before and 4-48h after irradiation with equi-erythemogenic doses of 311 and/or 290nm UVB (0.75-3.0 MED). A significant increase in the numbers of apoptotic epidermal cells (active-caspase-3+ cells) was seen after a single irradiation with 2-3 MEDs 311 nm UVB compared to 2-3 MEDs 290 nm UVB or untreated psoriasis (median 12/1000, 0/1000 and 0/1000 epidermal cells respectively; p<0.001). Routine clinical doses of 311nm also induced apoptosis. Immunochemistry and electron microscopy showed that the vast majority of apoptotic cells were keratinocytes. Live cell imaging of irradiated cultured keratinocytes and mathematical modelling showed that the rate of keratinocyte apoptosis observed in vivo was sufficient to account for clearance of psoriasis. Gene array analysis was used to examine differential gene regulation at these early time-points, prior to clinical response. Apoptosis and control of cell cycle pathways were affected by 311nm but not 290nm UVB. CDKN1A (WAF1/p21) showed the greatest fold change (27 fold up-regulation) following irradiation with 311nm UVB but not 290nm. Together these data provide evidence that epidermal keratinocyte apoptosis is a key mechanism in psoriasis resolution and identifies keratinocyte apoptosis as a potential target for future drug development.1 page(s

De Novo Mutations in MLL Cause Wiedemann-Steiner Syndrome

Excessive growth of terminal hair around the elbows (hypertrichosis cubiti) has been reported both in isolation and in association with a variable spectrum of associated phenotypic features. We identified a cohort of six individuals with hypertrichosis cubiti associated with short stature, intellectual disability, and a distinctive facial appearance, consistent with a diagnosis of Wiedemann-Steiner syndrome (WSS). Utilizing a whole-exome sequencing approach, we identified de novo mutations in MLL in five of the six individuals. MLL encodes a histone methyltransferase that regulates chromatin-mediated transcription through the catalysis of methylation of histone H3K4. Each of the five mutations is predicted to result in premature termination of the protein product. Furthermore, we demonstrate that transcripts arising from the mutant alleles are subject to nonsense-mediated decay. These findings define the genetic basis of WSS, provide additional evidence for the role of haploinsufficency of histone-modification enzymes in multiple-congenital-anomaly syndromes, and further illustrate the importance of the regulation of histone modification in development

Functional complementation studies identify candidate genes and common genetic variants associated with ovarian cancer survival

Common germline genetic variation and/or somatic alterations in tumours may be associated with survival in women diagnosed with ovarian cancer. The successful identification of genetic associations relies on a suitable strategy for identifying and testing candidate genes. We used microcell-mediated chromosome transfer approach and expression microarray analysis to identify genes that were associated with neoplastic suppression in ovarian cancer cell lines. Sixty-five tagging single nucleotide polymorphisms (tSNPs) in nine candidate genes were genotyped in similar to 1700 invasive ovarian cancer cases to look for associations with survival. For two of these genes, loss of heterozygosity (LOH) analysis of tSNPs in 314 ovarian tumours was used to identify associations between somatic gene deletions and survival. We identified significant associations with survival for a tSNP in caspase 5 (CASP5) [hazard ratio (HR) = 1.13 (95% CI: 1.00-1.27), P = 0.042] and two tSNPs in the retinoblastoma binding protein (RBBP8) gene [HR = 0.85 (95% CI: 0.75-0.95), P = 0.007 and HR = 0.83 (95% CI: 0.71-0.95), P = 0.009]. After adjusting for multiple prognostic factors in a multivariate Cox regression analysis, both associations in RBBP8 remained significant (P = 0.028 and 0.036). We then genotyped 314 ovarian tumours for several tSNPs in CASP5 and RBBP8 to identify gene deletions by LOH. For RBBP8, 35% of tumours in 101 informative cases showed somatic allelic deletion; LOH of RBBP8 was associated with a significantly worse prognosis [HR = 2.19 (95% CI: 1.36-3.54), P = 0.001]. In summary, a novel in vitro functional approach in ovarian cancer cells has identified RBBP8 as a gene for which both germline genetic variation and somatic alterations in tumours are associated with survival in ovarian cancer patients