Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the "physiologically" elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by "natural" infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3 deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.

BackgroundPancreatic cancer is a deadly disease. Discovery of the mutated genes that cause the inherited form(s) of the disease may shed light on the mechanism(s) of oncogenesis. Previously we isolated a susceptibility locus for familial pancreatic cancer to chromosome location 4q32–34. In this study, our goal was to discover the identity of the familial pancreatic cancer gene on 4q32 and determine the function of that gene.Methods and FindingsA customized microarray of the candidate chromosomal region affecting pancreatic cancer susceptibility revealed the greatest expression change in palladin (PALLD), a gene that encodes a component of the cytoskeleton that controls cell shape and motility. A mutation causing a proline (hydrophobic) to serine (hydrophilic) amino acid change (P239S) in a highly conserved region tracked with all affected family members and was absent in the non-affected members. The mutational change is not a known single nucleotide polymorphism. Palladin RNA, measured by quantitative RT-PCR, was overexpressed in the tissues from precancerous dysplasia and pancreatic adenocarcinoma in both familial and sporadic disease. Transfection of wild-type and P239S mutant palladin gene constructs into HeLa cells revealed a clear phenotypic effect: cells expressing P239S palladin exhibited cytoskeletal changes, abnormal actin bundle assembly, and an increased ability to migrate.ConclusionsThese observations suggest that the presence of an abnormal palladin gene in familial pancreatic cancer and the overexpression of palladin protein in sporadic pancreatic cancer cause cytoskeletal changes in pancreatic cancer and may be responsible for or contribute to the tumor's strong invasive and migratory abilities.

S100P is a member of the S100 family of calcium-binding proteins and there have been several recent reports of its overexpression in pancreatic ductal adenocarcinoma (PDAC). We have used Far Western screening and in vitro interaction assays to identify and confirm a novel target protein for S100P. We have named this protein S100PBPR, and shown that its interaction with S100P is dependent on Ca(2+) or Mg(2+). S100PBPR was found to localize to cell nuclei where S100P is also present, and the two proteins co-immunoprecipitate. By in situ hybridization, S100PBPR transcript was found in islet cells but not duct cells of the healthy pancreas. Both S100P and S100PBPR were detected by quantitative real-time polymerase chain reaction in pancreatic intraepithelial neoplasia (PanIN) and PDAC samples, and in situ hybridization revealed the presence of S100PBPR transcript in malignant PDAC cells. These data suggest that an interaction between S100P and S100PBPR may be involved in early pancreatic cancer. S100P was further investigated in PanIN lesions and immunohistochemical analysis showed its expression to correlate significantly with increasing grade of PanINs, being found as early as PanIN-1 with more prevalent expression in PanIN-2 and -3. These data suggest that S100P can be added to the genetic progression model for PDAC.

Up-regulation of S100P, a member of the S100 calcium-binding protein family, is an early molecular event in the development of pancreatic cancer and it is expressed at high levels in both precursor lesions and invasive cancer. To gain more insight into the molecular mechanisms underlying the functional roles of this protein, we stably overexpressed S100P in the Panc1 pancreatic cancer cell line and identified the consequent changes in global protein expression by twodimensional difference in-gel electrophoresis. The observed changes in target proteins were confirmed by Western blot analysis and immunofluorescence, whereas their functional effect was investigated using motility and invasion assays. In this study, we have shown that overexpression of S100P led to changes in the expression levels of several cytoskeletal proteins, including cytokeratins 8, 18, and 19. We have also shown disorganization of the actin cytoskeleton network and changes in the phosphorylation status of the actin regulatory protein cofilin. Additionally, we have shown that overexpression of S100P leads to increased expression of another early pancreatic cancer marker, S100A6, as well as the aspartic protease cathepsin D, both of which are involved in cellular invasion. Functional studies showed that the increased invasive potential of S100P-overexpressing cells was at least partially due to the increase in cathepsin D expression. In summary, our data suggest that these changes could contribute to the metastatic spread of pancreatic cancer and may explain the devastating prognosis of this disease. [Cancer Res 2007;67(18):8633-42]

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S-phase kinase associated protein 2 (Skp2) is a member of the F-box family of substrate recognition subunits of SCFubiquitin ligase complexes and controls progression from G 1 -S-phase by targeting cell cycle regulators such as p21 and p27. Its locus is at 5p13, a region of frequent amplification in several cancers including carcinoma of the cervix (CaCx). Overexpression of Skp2 has been observed in many cancers of an advanced stage. We examine the expression of Skp2 in 42 invasive CaCx and its correlation with tumour differentiation state and p27 expression. Using immunohistochemistry we found increased nuclear expression of Skp2 in 55% of invasive CaCx cases analysed. It is significant that poorly differentiated tumours invariably exhibit high Skp2 expression (>40% positive nuclei), whereas well-differentiated tumours express Skp2 at a lower level (<20% positive nuclei). Skp2 expression in normal cervical epithelia is <10% (positive nuclei). Increased Skp2 protein levels did not correlate inversely with p27 expression. Our data suggest that Skp2 may contribute to the progression of CaCx, however, unlike nonhuman papillomavirus (HPV) containing tumours, p27 is unlikely to be the major target protein contributing to malignant progression. The high prevalence of HPV types in CaCx may circumvent the need for Skp2 to eliminate p27.

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AIMSTo compare gastric acid suppression by netazepide, a gastrin/CCK2 receptor antagonist, with that by a proton pump inhibitor (PPI), and to determine if netazepide can prevent the trophic effects of PPI-induced hypergastrinaemia. METHODSThirty healthy subjects completed a double-blind, randomized, parallel group trial of oral netazepide and rabeprazole, alone and combined, once daily for 6 weeks. Primary end points were: basal and pentagastrin-stimulated gastric acid and 24 h circulating gastrin and chromogranin A (CgA) at baseline, start and end of treatment, gastric biopsies at baseline and end of treatment and basal and pentagastrin-stimulated gastric acid and dyspepsia questionnaire after treatment withdrawal. RESULTSAll treatments similarly inhibited pentagastrin-stimulated gastric acid secretion. All treatments increased serum gastrin, but the combination and rabeprazole did so more than netazepide alone. The combination also reduced basal acid secretion. Rabeprazole increased plasma CgA, whereas netazepide and the combination reduced it. None of the biopsies showed enterochromaffin-like (ECL) cell hyperplasia. Withdrawal of treatments led neither to rebound hyperacidity nor dyspepsia. CONCLUSIONSNetazepide suppressed pentagastrin-stimulated gastric acid secretion as effectively as did rabeprazole. The reduction in basal acid secretion and greater increase in serum gastrin by the combination is consistent with more effective acid suppression. Despite our failure to show rabeprazole-induced ECL cell hyperplasia and rebound hyperacidity, the increase in plasma CgA after rabeprazole is consistent with a trophic effect on ECL cells, which netazepide prevented. Thus, netazepide is a potential treatment for the trophic effects of hypergastrinaemia and, with or without a PPI, is a potential treatment for acid-related conditions. WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT• Gastrin controls gastric acid secretion and mucosal cell growth, especially of enterochromaffin-like (ECL) cells, which express gastrin/CCK2 receptors and release chromogranin A (CgA) into the circulation when stimulated by gastrin. • In non-clinical studies, acid suppression by a proton pump inhibitor (PPI) causes hypergastrinaemia which results in ECL cell growth and, after PPI withdrawal, rebound hyperacidity. Netazepide is an orally active, selective gastrin/CCK2 receptor antagonist, which suppresses acid production and prevents the trophic effects of PPI-induced hypergastrinaemia.• In healthy subjects, oral netazepide causes dose-dependent, persistent inhibition of gastric acid secretion, which leads to increased serum gastrin. WHAT THIS STUDY ADDS• In healthy subjects, netazepide and the PPI rabeprazole were similarly effective at suppressing pentagastrin-stimulated gastric acid secretion and increasing serum gastrin.• Rabeprazole increased plasma CgA, a sign of ECL cell hyperactivity, whereas netazepide reduced plasma CgA, a sign of ECL cell hypoactivity. Netazepide also prevented the increase in CgA resulting from rabeprazole-induced hyp...