George C. Mayne

Induction of cancer cell apoptosis by α-tocopheryl succinate: molecular pathways and structural requirements

The vitamin E analog α‐tocopheryl succinate (α‐TOS) can induce apoptosis. We show that the proapoptotic activity of α‐TOS in hematopoietic and cancer cell lines involves inhibition of protein kinase C (PKC), since phorbol myristyl acetate prevented α‐TOS‐triggered apoptosis. More selective effectors indicated that α‐TOS reduced PKCα isotype activity by increasing protein phosphatase 2A (PP2A) activity. The role of PKCα inhibition in α‐TOS‐induced apoptosis was confirmed using antisense oligonucleotides or PKCα overexpression. Gain‐ or loss‐of‐function bcl‐2 mutants implied modulation of bcl‐2 activity by PKC/ PP2A as a mitochondrial target of α‐TOS‐induced proapoptotic signals. Structural analogs revealed that α‐tocopheryl and succinyl moieties are both required for maximizing these effects. In mice with colon cancer xenografts, α‐TOS suppressed tumor growth by 80%. This epitomizes cancer cell killing by a pharmacologically relevant compound without known side effects.—Neuzil, J., Weber, T., Schröder, A., Lu, M., Ostermann, G., Gellert, N., Mayne, G. C., Olejnicka, B., Nègre‐Salvayre, A., Stícha, M., Coffey, R. J., Weber, C. Induction of cancer cell apoptosis by α‐tocopheryl succinate: molecular pathways and structural requirements. FASEB J. 15, 403‐415 (2001)

Ceramide induces apoptosis in PC12 cells

The novel lipid second messenger, ceramide, induced apoptosis in PC12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous C2‐ceramide in a dose‐ and time‐dependent manner. Natural ceramide and C6‐ceramide had a similar effect. This response was specific since the structural analog C2‐dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin‐1β converting enzyme (ICE)‐like proteases, Z‐VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE‐like protease activation in ceramide‐induced apoptosis in PC12 cells.

Evidence That Protein Kinase Cε Mediates Phorbol Ester Inhibition of Calphostin C- and Tumor Necrosis Factor-α-induced Apoptosis in U937 Histiocytic Lymphoma Cells

Protein kinase C (PKC) activators, such as the tumor-promoting phorbol esters, have been reported to protect several cell lines from apoptosis induced by a variety of agents. Recent evidence suggests that PKCε is involved in protection of cardiac myocytes from hypoxia-induced cell death (Gray, M. O., Karliner, J. S., and Mochly-Rosen, D. (1997) J. Biol. Chem.272, 30945–30951). We investigated the protective effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on U937 histiocytic lymphoma cells induced to undergo apoptosis by tumor necrosis factor-α (TNF-α) or by the specific PKC inhibitor calphostin C. U937 cells were transiently permeabilized with a peptide (εV1-2) derived from the V1 region of PKCε that has been reported to specifically block translocation of PKCε. The εV1-2 peptide blocked the inhibitory effect of TPA on both TNF-α- and calphostin C-induced apoptosis. A scrambled version of εV1-2 and a peptide reported to inhibit PKCβ translocation (βC2-4) had no effect on the ability of TPA to inhibit apoptosis. These results suggest that PKCε is required for the protective effect of TPA in TNF-α- and calphostin C-induced apoptosis. Furthermore, calphostin C reduced membrane-associated PKCε activity and immunoreactivity, suggesting that PKCε may play an important role in leukemic cell survival.

Cost-effectiveness of endoscopic surveillance of non-dysplastic Barrett's esophagus

BACKGROUND Endoscopic surveillance for non-dysplastic Barretts esophagus (BE) is contentious and its cost effectiveness unclear. OBJECTIVE To perform an economic analysis of endoscopic surveillance strategies. DESIGN Cost-utility analysis by using a simulation Markov model to synthesize evidence from large epidemiologic studies and clinical data for surveillance, based on international guidelines, applied in a coordinator-managed surveillance program. SETTING Tertiary care hospital, South Australia. PATIENTS A total of 2040 patient-years of follow-up. INTERVENTION (1) No surveillance, (2) 2-yearly endoscopic surveillance of patients with non-dysplastic BE and 6-monthly surveillance of patients with low-grade dysplasia, (3) a hypothetical strategy of biomarker-modified surveillance. MAIN OUTCOME MEASUREMENTS U.S. cost per quality-adjusted life year (QALY) ratios. RESULTS Compared with no surveillance, surveillance produced an estimated incremental cost per QALY ratio of

MicroRNA-143 and -205 Expression in Neosquamous Esophageal Epithelium Following Argon Plasma Ablation of Barrett’s Esophagus

60,858. This was reduced to

miR-200 family expression is downregulated upon neoplastic progression of Barrett’s esophagus

38,307 when surveillance practice was modified by a hypothetical biomarker-based strategy. Sensitivity analyses indicated that the likelihood that surveillance alone was cost-effective compared with no surveillance was 16.0% and 60.6% if a hypothetical biomarker-based strategy was added to surveillance, at an acceptability threshold of

Centrifugation facilitates transduction of green fluorescent protein in human monocytes and macrophages by adenovirus at low multiplicity of infection

100,000 per QALY gained. LIMITATIONS Treatment options for BE that overlap those for symptomatic GERD were omitted. CONCLUSION By using best available estimates of the malignant potential of BE, endoscopic surveillance of patients with non-dysplastic BE is unlikely to be cost-effective for the majority of patients and depends heavily on progression rates between dysplasia grades. However, strategies that modify surveillance according to cancer risk might be cost-effective, provided that high-risk individuals can be identified and prioritized for surveillance.

MicroRNAs and esophageal cancer--implications for pathogenesis and therapy.

IntroductionAblation of Barrett’s esophagus using Argon plasma coagulation (APC) is usually followed by the formation of a neosquamous epithelium. Investigating simple columnar or stratified squamous epithelium associated cytokeratin and microRNA (miRNA) expression in neo-squamous epithelium could help determine the identity and stability of the neosquamous epithelium.MethodsNine patients underwent ablation of Barrett’s esophagus with APC. Biopsies were collected from Barrett’s esophagus mucosa and proximal normal squamous epithelium before ablation, and from neosquamous and normal squamous epithelium after ablation. Additional esophageal mucosal biopsies from ten nonrefluxing subjects were used as a reference. RNA was extracted and real-time polymerase chain reaction was used to measure the expression of the cytokeratins CK-8 and CK-14 and the microRNAs miR-143 and miR-205.ResultsCK-8 and miR-143 expression were significantly higher in Barrett’s esophagus mucosa, compared to neosquamous and normal squamous epithelium before and after APC, whereas miRNA-205 and CK-14 expression was significantly lower in Barrett’s esophagus mucosa compared to all categories of squamous mucosa. The expression of CK-8, CK-14, miR-205, and miR-143 was similar between neosquamous epithelium compared to normal squamous epithelium in patients with Barrett’s esophagus. Only miR-143 expression was significantly higher in neosquamous and normal squamous epithelium before and after APC compared to normal squamous epithelium from control subjects (p < 0.004).ConclusionsThe expression levels of cytokeratins and miRNAs studied in post-ablation neosquamous epithelium and normal squamous epithelium in patients with Barrett’s esophagus are similar. In patients with Barrett’s esophagus, miR-143 expression is still elevated in both neosquamous mucosa, and the squamous mucosa above the metaplastic segment, suggesting that this mucosa may not be normal; i.e., it is different to that seen in subjects without Barrett’s esophagus. miR-143 could promote a Barrett’s epithelium gene expression pattern, and this could have a role in development of Barrett’s esophagus.

Galanin mediates the pathogenesis of cerulein-induced acute pancreatitis in the mouse.

AIM To investigate miR-200 family expression in Barretts epithelium, gastric and duodenal epithelia, and esophageal adenocarcinoma. METHODS Real-time reverse transcriptase-polymerase chain reaction was used to measure miR-200, ZEB1 and ZEB2 expression. Ingenuity Pathway Analysis of miR-200 targets was used to predict biological outcomes. RESULTS Barretts epithelium expressed lower levels of miR-141 and miR-200c than did gastric and duodenal epithelia (P < 0.001). In silico analysis indicated roles for the miR-200 family in molecular pathways that distinguish Barretts epithelium from gastric and duodenal epithelia, and which control apoptosis and proliferation. All miR-200 members were downregulated in adenocarcinoma (P < 0.02), and miR-200c expression was also downregulated in non-invasive epithelium adjacent to adenocarcinoma (P < 0.02). The expression of all miR-200 members was lower in Barretts epithelium derived high-grade dysplastic cell lines than in a cell line derived from benign Barretts epithelium. We observed significant inverse correlations between miR-200 family expression and ZEB1 and ZEB2 expression in Barretts epithelium and esophageal adenocarcinoma (P < 0.05). CONCLUSION miR-200 expression might contribute to the anti-apoptotic and proliferative phenotype of Barretts epithelium and regulate key neoplastic processes in this epithelium.

Healthcare resource use and medical costs for the management of oesophageal cancer

Due to their phagocytic and poorly proliferative nature, it has been difficult to transfect human monocytes and macrophages. Adenoviral vectors have recently allowed transduction of a high percentage of human macrophages, but only after CSF upregulation of the integrins, alphavbeta3 or alphavbeta5, during culture for 48 h, a time allowing significant monocyte to macrophage differentiation. In our hands, after 24-h incubation with M-CSF (20 ng/ml) and a further 24-h incubation with an adenoviral vector encoding green fluorescent protein (AdV-GFP) [multiplicity of infection (MOI)=50:1], only 35% of CD14-positive cells express GFP. We report that centrifugation of these cells with AdV-GFP at 2000 x g for 1 h at 37 degrees C significantly enhanced the number of cells expressing GFP (to 65%) and the level of GFP expression per transduced cell (fivefold). The viability of the cells was not compromised (<5 % CD14-positive cells were 7-aminoactinomycin D (7AAD)-positive after 24 h AdV-GFP exposure at MOI=50:1). Centrifugation allowed efficient transduction of monocytes and macrophages with an MOI at least tenfold lower than otherwise required and did not activate the transduced cells or affect their ability to produce TNFalpha or IL-1beta in response to lipopolysaccharide (LPS). This methodology was also suitable for transducing large numbers of in vitro monocyte-derived macrophages (MDMac) and macrophages isolated from synovial fluids with up to 75-80% of CD14-positive cells transduced after 24-h exposure to AdV-GFP (50:1) and centrifugation (2000 x g). This methodology should provide significant expression of transgenes in human monocytes and macrophages.