Considerable progress has been made recently in our understanding of the role of ceramide in the induction of apoptotic cell death. Ceramide is produced by cancer cells in response to exposure to radiation and most chemotherapeutics and is an intracellular second messenger that activates enzymes, leading to apoptosis. Because of its central role in apoptosis, pharmacologic manipulation of intracellular ceramide levels should result in attenuation or enhancement of drug resistance. This may be achieved through direct application of sphingolipids or by the inhibition/activation of the enzymes that either produce or use ceramide. In addition, attention should be given to the subcellular location of ceramide generation, because this has been shown to affect the biological activity of sphingolipids. This review summarizes the sphingolipid biosynthetic pathway, as it relates to the identification of important targets for drug discovery, and the development of novel agents capable of enhancing chemotherapy. [Mol Cancer Ther 2006;5(2):200 -8]

At a cutoff of 10.2 units/mL, 41 (77%) of 53 pancreatic cancer patients, none of the healthy individuals (n = 43), and only four (5%) of 87 patients with pancreatitis were positive above this value. Among nonpancreatic cancers investigated, colorectal cancers gave the highest percentage of positives (14%; five of 36). Overall, the sensitivity and specificity of the immunoassay for pancreatic cancer were 77% and 95%, respectively. Receiver operator characteristic analyses for discrimination of pancreatic cancer from pancreatitis provided an area under the curve of 0.89 (95% CI, 0.82 to 0.93), with a specificity of 95.4% and a positive likelihood ratio of 16.8. A direct pair-wise comparison of PAM4 and CA19-9 immunoassays for discrimination of pancreatic cancer and pancreatitis resulted in a significant difference (P < .003), with the PAM4 immunoassay demonstrating superior sensitivity and specificity. CONCLUSION The high sensitivity and specificity observed suggest that the PAM4-based immunoassay of circulating MUC1 may be useful in the diagnosis of pancreatic cancer.

Purpose: The anti-MUC1monoclonal antibody (MAb), PAM4, has a high specificity for pancreatic adenocarcinoma compared with other cancers, normal tissues, or pancreatitis. In order to assess its role in early pancreatic cancer development, we examined the expression of the PAM4-reactive MUC1 in the noninvasive precursor lesions, pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN). Most patients with pancreatic cancer do not develop symptoms until after the disease has metastasized. The early detection and diagnosis of pancreatic cancer, as well as appropriate staging of the disease, would almost certainly provide a survival advantage. With few options currently available, there remains a critical need for the development of procedures for the accurate detection of early pancreatic cancer. A growing body of evidence supports the view that pancreatic adenocarcinoma can arise from histologically well-defined noninvasive lesions within the pancreatic ducts (1 -3). These precursor lesions include pancreatic intraepithelial neoplasias (PanIN), and the larger intraductal papillary mucinous neoplasms (IPMN) and mucinous cystic neoplasms (3). These precursors are true neoplasms that share many of the aberrant genetic alterations and expression of biomarkers characteristic of invasive adenocarcinoma (4 -8). It has been proposed that PanIN lesions progress from the early PanIN-1A (flat epithelium with minimal atypia) and PanIN-1B (papillary epithelium with minimal atypia) to PanIN-2 (papillary epithelium with moderate atypia) to PanIN-3 (papillary epithelium with marked atypia), and eventually invasive adenocarcinomas.In an effort to improve the early detection of pancreatic neoplasia, several investigators have examined PanIN lesions for specific genetic and protein biomarkers. These include survivin (9), Her2neu (10, 11), mesothelin (2), p53 (2), DPC4 (2, 12), and several mucin species (MUC1, MUC4, MUC5AC, etc. refs. 2,6,(13)(14)(15) among others. For the most part, detection of these biomarkers has been shown by the use of immunohistochemical labeling of tissues. Certain biomarkers (e.g., mucins) are highly complex molecules that may, or may not, display specific epitope structures at varying stages in the progression from PanIN-1 to invasive adenocarcinoma. Indeed, discrepancies are apparent when MUC1 is identified by the use of monoclonal antibodies (MAb) that react with the variable number of tandem repeats (VNTR) core peptide versus MAbs reactive with glycosylated structures. MUC1 may be reported as present or absent from normal tissues, precursor lesions, and/or adenocarcinoma based on the specific MAb being used. Thus, caution is needed in determining which events (i.e., expression

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Background: Pancreatic adenocarcinoma is an almost universally lethal disease, in large part, due to our inability to detect early-stage disease. Monoclonal antibody PAM4 is reactive with a unique biomarker expressed by >85% of pancreatic adenocarcinomas. In this report, we examined the ability of a PAM4-based immunoassay to detect early-stage disease.Materials and Methods: The PAM4-based immunoassay was used to quantitate antigen in the serum of healthy volunteers (n = 19), patients with known pancreatic adenocarcinoma (n = 68), and patients with a primary diagnosis of chronic pancreatitis (n = 29).Results: Sensitivity for detection of pancreatic adenocarcinoma was 82%, with a false-positive rate of 5% for healthy controls. Patients with advanced disease had significantly higher antigen levels than those with early-stage disease (P < 0.01), with a diagnostic sensitivity of 91%, 86%, and 62% for stage 3/stage 4 advanced disease, stage 2, and stage 1, respectively. We also evaluated chronic pancreatitis sera, finding 38% positive for antigen; however, this was discordant with immunohistochemical findings that suggest the PAM4 antigen is not produced by inflamed pancreatic tissue. Furthermore, several of the serum-positive pancreatitis patients, for whom tissue specimens were available for pathologic interpretation, had evidence of neoplastic precursor lesions.Conclusions: These results suggest the use of the PAM4 serum assay to detect early-stage pancreatic adenocarcinoma and that positive levels of PAM4 antigen are not derived from inflamed pancreatic tissues but rather may provide evidence of subclinical pancreatic neoplasia.Effect: The ability to detect pancreatic adenocarcinoma at an early stage could provide for early therapeutic intervention with potentially improved patient outcomes.

We have examined the mechanism by which sphingomyelin (SM) enhances chemotherapy in human pancreatic cancer cells, focusing on the correlation between ceramide metabolism and apoptosis. Dose response curves for gemcitabine in the absence or presence of 0.2 mg/mL SM provided IC 50 values of 78.3 ؎ 13.7 and 13.0 ؎ 3.0 nmol/L, respectively. The cytotoxic effect of the combined treatment was synergistic (combination index ‫؍‬ 0.36). Using annexin-V staining, the percentage of apoptotic cells was 3.6 ؎ 2.6% for the untreated cells, 6.5 ؎ 3.8% for the 0.2 mg/mL SM-treated cells, and 19.9 ؎ 12.9% for the 100 nmol/L gemcitabinetreated cells, but increased significantly to 42.1 ؎ 12.7% with the combined treatment (P < 0.001, compared with gemcitabine-treated group). The percentage of cells losing mitochondrial membrane potential followed a similar trend. The ceramide content of untreated and gemcitabinetreated cells was not significantly different (0.46 ؎ 0.29 and 0.59 ؎ 0.34 pmol ceramide/nmole PO 4 ). However, when 0.2 mg/mL SM was added, ceramide levels were 1.09 ؎ 0.42 and 1.58 ؎ 0.55 pmol ceramide/nmol PO 4 , for the SM alone and SM with gemcitabine-treated cells, respectively (P ‫؍‬ 0.038). Acidic SMase was activated by exposure to gemcitabine but not SM, whereas the activities of neutral SMase and glycosylceramide synthase did not change with either gemcitabine or SM. The data are consistent with gemcitabine-induced activation of acidic SMase and indicate that the addition of SM can yield increased production of ceramide, mitochondrial depolarization, apoptosis, and cell death. Because SM by itself is relatively nontoxic, addition of this lipid to agents that induce apoptosis may prove useful to enhance apoptosis and increase cytotoxicity in cancer cells.

Bioactive sphingolipids are potent intracellular signaling molecules having profound effects on cell death, growth, and differentiation. Pharmacologic manipulation of sphingolipid levels could have a significant effect on the induction of apoptosis by anticancer agents, and thus, improve treatment efficacy. We observed that gemcitabine cannot completely kill AsPc1 and Panc1 human pancreatic cancer cells in culture; even at high concentrations of gemcitabine, 30% to 40% of the cells remain viable. By adding sphingomyelin to the culture medium, gemcitabineinduced cell death increased synergistically to >90%. Panc1 cells that survived high concentrations of gemcitabine had an increase in β-galactosidase activity, a marker of senescence. The inclusion of sphingomyelin with gemcitabine reduced β-galactosidase activity, as compared with cells treated with gemcitabine alone. Expression of p21 waf1/cip1 in both cell lines exposed to sphingomyelin, gemcitabine, and gemcitabine + sphingomyelin varied relative to the untreated group. C 8 -ceramide induced both cell death and senescence in a dose-dependent manner. These results indicate that gemcitabine induces senescence in pancreatic cancer cells and that sphingomyelin-enhanced chemosensitivity is achieved through reducing the induction of senescence by redirecting the cell to enter the apoptotic pathway. Ceramide levels seem to be critical to this decision, with cell cycle progression being uninhibited at low ceramide levels, senescence induced at moderate levels, and apoptosis initiated at high levels. Our results provide further evidence that targeting the sphingolipid metabolism is a means of enhancing the efficacy of chemotherapeutic agents. (Mol Cancer Res 2009;7(6):890-6)

The function of transcription termination factor rho from Escherichia coli is dependent upon its ability to bind RNA. To delineate the extent of the RNA-binding domain in the rho polypeptide, plasmid-borne copies of altered forms of the rho gene were expressed to yield truncated versions. These proteins were then isolated and assayed for their ability to bind an RNA oligonucleotide [oligo(C)8] using an ultraviolet light-induced cross-linking assay. A fragment consisting of the first 116 amino acid residues, rho(1-116), bound oligo(C)8 with nearly the same affinity and specificity as the intact protein. Smaller derivatives lacking 5, 13, or 22 residues from the N terminus or with 2 fewer residues at the C terminus bound RNA with reduced affinity, while derivatives lacking 27 N-terminal residues or having just the first 109 residues were unable to bind RNA. Derivatives lacking N-terminal residues were considerably less soluble than rho(1-116). The physical properties of rho(1-116) indicate that it possesses approximately 20% each of alpha-helix and beta-sheet and is monomeric in solution. Thus, the results show that this fragment, which contains an RNP1 sequence motif, will be a good model for future physical-chemical studies of the protein-RNA interactions of rho.