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Objective: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. Design: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. Results: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. Conclusion: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models., SCOPUS: ar.j, info:eu-repo/semantics/published

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8(-/-)) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8(-/-) beta cells and were replaced by immature, pale insulin "progranules,'' which were larger than in ZnT8(+/+) islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8(-/-) mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8(-/-) genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

OBJECTIVE: In beta cells, zinc co-crystallises with insulin in dense-core secretory granules, but the exact role of zinc in insulin biosynthesis, storage and secretion is unknown. Polymorphism in the ZnT8 gene confers susceptibility to T2D, and autoantibodies to ZnT8 protein are observed in T1D. The aim of this study was to assess the role of ZnT8 in beta cells using mice that are deficient in ZnT8 expression (ZnT8-/- mice). MATERIALS AND METHODS: ZnT8-/- mice have a deletion of the promotor region and first exon. ZnT8 expression was assessed by RT-PCR, Western blots, and immunohistochemistry. Beta cell ultra-structure was investigated by transmission electron microscopy. Glucose tolerance was measured at age 6, 12, 25 and 52 weeks and after 10 weeks on high fat diet. Insulin processing was analysed via 35S pulse chase experiments and in situ mass spectral tissue profiling of pancreatic islets. Insulin release from isolated islets was measured in perifusion experiments, and zinc exocytosis from islet cell clusters was monitored by total internal fluorescence microscopy. RESULTS: ZnT8-/- mice were completely negative for the expression of islet ZnT8 mRNA and protein. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, indicating that ZnT8 is the only zinc transporter responsible for influx of zinc into the secretory granules. Also dense-core insulin granules were absent in ZnT8-/- beta cells (92.2 ± 6.6 vs 7.0 ± 2.7 %, p<0.0001); instead, immature, pale insulin “progranules” were detected, which were larger (433±83 vs 375±84 nm, p<0.001) than insulin granules in control cells. This phenotypic change was associated with loss of in situ and in vitro staining of islets with dithizone and disappearance of the bright light reflection of isolated islets. In contrast, rates of insulin biosynthesis, insulin content and insulin release after glucose stimulation were well preserved in ZnT8-/- mice. Moreover, when ZnT8-/- mice were fed a norma

Aims: To investigate the correlation between O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and benefit from temozolomide in patients with recurrent high-grade glioma. Patients and methods: A real-time, quantitative, methylation-specific PCR assay was performed on archival tissue blocks from patients treated with temozolomide at the first recurrence. Results: A subgroup of 38 patients who were chemotherapy-naive at recurrence was analysed (22 glioblastoma, 12 anaplastic astrocytoma [AA] and 4 anaplastic oligoastrocytoma [AOA]); none had 1p/19q loss. Among 10 (26%) patients with a hypermethylated MGMT promoter, none experienced disease progression within the first two treatment cycles compared with 12 of 28 (43%) patients with an unmethylated promoter (p = 0.016). By Cox multivariate analysis, tumour grade and MGMT promoter methylation correlated with time to progression (p < 0.05); MGMT promoter methylation correlated with superior overall survival in AA/AOA but not in glioblastoma. Conclusions: MGMT promoter methylation predicted a survival benefit in patients with 1p/19q intact AA/AOA treated with temozolomide at recurrence. © 2008 Elsevier Ltd. All rights reserved., SCOPUS: ar.j, info:eu-repo/semantics/published