BLT2 is expressed in PanINs, IPMNs, pancreatic cancer and stimulates tumour cell proliferation.

Pancreatic cancer has an abysmal prognosis. Targets for early detection, prevention and therapy are desperately needed. Inflammatory pathways have an important impact on tumour growth and progression. Expression of BLT2 (the second leukotriene B(4) receptor) was investigated by real-time RT-PCR and immunohistochemistry. Cell proliferation was studied after stable transfection with BLT2, after treatment with siRNA and Compound A as an agonist. BLT2 is expressed in all pancreatic cancer cell lines. Results from real-time RT-PCR revealed significant overexpression of BLT2 in malignant intraductal papillary mucinous neoplasias (IPMNs) and pancreatic adenocarcinoma. Intense staining was evident in IPMNs, infiltrating tumour cells and advanced pancreatic intraepithelial neoplasias (PanINs) but not in normal ductal cells. Overexpression of BLT2 as well as stimulation of Colo357, Panc-1 and AsPC1 cells with Compound A caused a significant increase in tumour cell proliferation, an effect reversed after siRNA treatment. This study demonstrates for the first time the expression of BLT2 in the pancreas and overexpression in pancreatic cancers and malignant IPMNs in particular. Upregulation of BLT2 is already evident in precursor lesions (PanINs, IPMNs). Overexpression of this receptor leads to significant growth stimulation. Therefore, we suggest BLT2 as a new target for chemoprevention and therapy for pancreatic cancer.

Genome-wide identification of aberrantly methylated promoter associated CpG islands in acute lymphocytic leukemia.

We performed a genome-wide analysis of promoter associated CpG island methylation using methylated CpG island amplification (MCA) coupled to representational differential analysis (RDA) or a DNA promoter microarray in acute lymphoblastic leukemia (ALL). We identified 65 potential targets of methylation with the MCA/RDA approach, and 404 with the MCA/array. Thirty-six (77%) of the genes identified by MCA/RDA were shared by the MCA/array approach. Chromosomal location of these genes was evenly distributed in all autosomes. Functionally, 303 of these genes clustered in 18 molecular pathways. Of the 36 shared genes, 31 were validated and 26 were confirmed as being hypermethylated in leukemia cell lines. Expression analysis of eight of these genes was epigenetically modulated by hypomethylating agents and/or HDAC inhibitors in leukemia cell lines. Subsequently, DNA methylation of 15 of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) was validated in primary ALL samples. Patients with methylation of multiple CpG islands had a worse overall survival. This is the largest published list of potential methylation target genes in human leukemia offering the possibility of performing rational unbiased methylation studies in ALL.

12-lipoxygenase metabolite 12(S)-HETE stimulates human pancreatic cancer cell proliferation via protein tyrosine phosphorylation and ERK activation.

We previously reported that inhibition of the 12-lipoxygenase pathway abolished proliferation and induced apoptosis in several pancreatic cancer cell lines. Furthermore, the 12-lipoxygenase product 12(S)-HETE stimulated pancreatic cancer cell proliferation and reversed 12-lipoxygenase inhibitor-induced growth inhibition. We investigated the underlying mechanism for 12(S)-HETE-induced pancreatic cancer cell proliferation, using 2 human pancreatic cancer cell lines, PANC-1 and HPAF. Cell proliferation was monitored by both thymidine incorporation and cell number. Western blotting was used to investigate the effect of 12(S)-HETE on cellular protein tyrosine phosphorylation as well as ERK, P38 MAPK and JNK/SAPK phosphorylation. 12(S)-HETE markedly stimulated proliferation of pancreatic cancer cells in a time- and concentration-dependent manner. In parallel, 12(S)-HETE induced tyrosine phosphorylation of multiple cellular proteins, while inhibition of tyrosine kinase by genestein abolished 12(S)-HETE-induced proliferation, indicating that intracellular protein tyrosine kinase activation is involved in the mitogenic effects of 12(S)-HETE. Following treatment with 12(S)-HETE, both ERK and P38 MAPK, but not JNK/SAPK, were phosphorylated. The specific MEK inhibitors PD098059 and U0126, which in turn suppress ERK, abolished 12(S)-HETE-stimulated proliferation. In contrast, inhibition of P38 MAPK with SB203580 did not affect 12(S)-HETE-stimulated pancreatic cancer cell proliferation. Furthermore, 12(S)-HETE-stimulated ERK phosphorylation was inhibited by genestein, indicating that tyrosine phosphorylation is essential for ERK activation. These findings suggest that both ERK and cellular protein tyrosine kinase activation are involved in 12(S)-HETE-induced pancreatic cancer cell proliferation but P38 and JNK/SAPK are not involved in this mitogenic effect.

Cyclooxygenases and lipoxygenases as potential targets for treatment of pancreatic cancer.

Pancreatic adenocarcinoma is characterized by poor prognosis, late diagnosis and lack of response to conventional therapies. The incidence of this disease shows no sign of declining in the Western world. Thus, new targets need to be identified for pancreatic cancer treatment. In particular, new chemotherapeutic agents would be extremely beneficial for control of unresectable cancer and metastatic lesions as well as for prevention of this deadly disease. Mounting evidence suggests that both lipoxygenases (LOXs) and cyclooxygenases (COXs), the key enzymes for arachidonic acid metabolism, have a profound influence on the development and progression of several human cancers. Recent evidence suggests that both COX and LOX pathways are important in pancreatic cancer. Results from immunocytochemical, RT-PCR, and Western blotting studies have shown that COX, specifically COX-2, is upregulated in human pancreatic cancer cell lines as well as human pancreatic cancer tissues compared with normal ductal cells and normal pancreas specimens. Agents that block COX enzymes significantly inhibit pancreatic cancer growth both in vitro and in vivo, in parallel with induction of apoptosis. Expression of both 5-LOX and 12-LOX is also seen in pancreatic cancer, although compared to the expression of COX this has not been extensively investigated. Chemical inhibitors or antisense oligonucleotides that block either 5-LOX or 12-LOX cause marked inhibition of pancreatic cancer cell proliferation. On the other hand, LOX metabolites stimulate growth of the tumor cells and reverse LOX-inhibitor-induced growth inhibition, suggesting the specific role of LOX in regulating pancreatic cancer cell proliferation. Although questions still need to be answered, such as the underlying mechanisms for COX and LOX-induced growth inhibition, both COX and LOX pathways are potential targets for pancreatic cancer treatment and chemoprevention. COX and LOX enzyme inhibitors are available and have been shown to be relatively safe in the treatment of other diseases.

Blockade of cyclooxygenase-2 inhibits proliferation and induces apoptosis in human pancreatic cancer cells.

Cyclooxygenase (COX), also referred to as prostaglandin endoperoxide synthase, is a key enzyme in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Epidemiologic, animal and in vitro observations show a positive correlation between the expression of COX (especially COX-2) and colonic cancer development, growth and apoptosis. Constitutive expression of COX-2 in human pancreatic cancer cells was recently reported. To evaluate the potential role of COX in pancreatic cancer, RT-PCR was used to determine the constitutive expression of COX-2 in four pancreatic cancer cell lines. MiaPaCa2, PANC-1, HPAF, ASPC-1. The effect of COX blockade with either the general COX inhibitor, indomethacin, or the specific COX-2 inhibitor, NS-398, on [3H]-thymidine incorporation and cell number was investigated in these four pancreatic cancer cell lines. In addition, the effects of these COX inhibitors on pancreatic cancer cell apoptosis was evaluated by DNA propidium iodide staining and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. All four human pancreatic cancer cell lines expressed COX-2 and their proliferation was concentration- and time-dependently inhibited by both indomethacin andNS398. Substantial apoptosis was also induced by treatment of pancreatic cancer cells with either indomethacin or NS398, as indicated by both DNA propidium iodide staining and the TUNEL assay. Furthermore, indomethacin and NS398 were equipotent for growth inhibition and induction of apoptosis, indicating that eicosanoid synthesis via COX-2 is involved in pancreatic cancer cell proliferation and survival. In conclusion, these findings suggest that the COX pathway, especially COX-2, contributes to the growth and apoptosis of pancreatic cancer. Specific COX-2 inhibitors are likely to be valuable for the treatment and prevention of this deadly cancer.

Absorbance detection of amino acids by laser wave mixing in microbore liquid chromatography.

Nonlinear optical phase conjugation by degenerate four-wave mixing is demonstrated as a sensitive "absorbance" detection method for microbore high-performance liquid chromatography. An argon ion laser operating at the 488-nm line is used as the excitation light source to generate the wave-mixing signal for dabsyl-labeled amino acids. Advantages of the nonlinear laser detection method include: virtually 100% optical signal collection efficiency, generation of the signal in the form of a coherent laser beam, signal measurement against a virtually dark background, reliable detection of small absorbance values, excellent detection sensitivity for both fluorescing and non-fluorescing analytes, relatively simple one-color one-laser optical setup, and low power or energy requirements for continuous-wave or pulsed lasers. Using our one-laser one-color nonlinear laser detector for "absorbance" measurements in liquid chromatography, we report a crude preliminary "injected" detection limit of 780 fmol for glycine.

Sensitive absorbance detection method for capillary electrophoresis based on laser wave-mixing.

Forward-scattering four-wave mixing is demonstrated as a sensitive absorbance detection method for capillary electrophoresis, using an argon ion laser operating at 457.9 nm. Since this four-wave mixing laser technique utilizes only two input laser beams, it offers important advantages, including ease of optical alignment, high wave-mixing efficiency and low excitation power requirements. In addition, since the analytical signal is a laser-like coherent beam, highly efficient optical signal detection can be performed with minimum optical background noise. Excellent detection sensitivity and short absorption path lengths, and hence, small detector probe volumes, are some of the useful features this absorbance detection method offers for on-column detection of both fluorescing and non-fluorescing analytes in capillary electrophoresis and liquid chromatography. Preliminary "detected" concentration detection limit of 8.5.10(-8) M, mass detection limit of 13 amol and an absorbance-unit detection limit of 1.35.10(-5) AU are determined for dabsyl-glycine using this absorbance detection method.

Sensitive circular dichroism spectroscopy based on nonlinear degenerate four-wave mixing.

Degenerate four-wave mixing is demonstrated as an effective and sensitive laser analytical spectroscopic method for circular dichroism measurements. A forward-scattering degenerate four-wave mixing optical setup is used to obtain simple optical alignment, highly efficient wave mixing, and very effective use of low laser power. This nonlinear laser-based circular dichroism method offers many advantages, including easy and efficient optical signal collection, use of very short analyte path lengths (e.g., 0.1 mm), and excellent detection sensitivity that is comparable or better than conventional laser-based or non-laser-based circular dichroism methods. Using an analyte path length of only 0.1 mm, and a probe volume of 98 pL, a circular dichroism mass detection limit of 0.68 pg or 2.8 fmol is reported for (+)Co(en)3(3+).

Forward-scattering degenerate four-wave mixing as a simple sub-attomole-sensitive nonlinear laser analytical spectrometric method.

Optical phase conjugation by "forward-scattering" degenerate four-wave mixing in an absorbing liquid analyte solution is reported as a sensitive and simple nonlinear laser spectroscopic method. Since only two input laser beams are used in this nonlinear four-wave mixing setup, it offers important advantages including ease of optical alignment, efficient use of input photon density, low laser power requirements, and high wave-mixing efficiency. In addition, since the phase-conjugate signal is a laser beam, optical signal detection is very efficient and the signal-to-noise is excellent. Important characteristics of this novel nonlinear laser technique, including signal dependence on analyte concentration, individual input beam power, and modulation frequencies, are examined. Excellent detection sensitivity, small detection volume, and convenient sample introduction promise many applications for this nonlinear laser spectroscopic method. Preliminary detection limits of 0.7 amol of eosin B and 45 amol of iodine inside a probe volume of 98 pL are reported using a forward-scattering degenerate four-wave mixing setup.

Trace-concentration detection of cobalt in a liquid flow cell by degenerate four-wave mixing using low-power off-resonant laser excitation.

Optical phase conjugation by degenerate four-wave mixing (D4WM) in an absorbing metal-ion solution using a low-power argon-ion laser as the excitation source is demonstrated. This nonlinear laser technique can be used as a sensitive analytical spectroscopic method for trace-concentration measurement of metal ions in a small-volume continuously flowing analyte cell. Several important characteristics are discussed, including the effects of solvent properties, excitation wave-length, laser intensity, and analyte absorptivity on signal intensity. Detection of 0.26 ng (4.4 pmol) of cobalt inside the laser probe volume of 0.14 microL is reported using an excitation wavelength that is 136 nm away from the maximum absorption wavelength of the analyte solution. The minimum absorbance measured in our D4WM experiment is 2.0 X 10(-5) without complex formation for cobalt. The D4WM detection sensitivity, in terms of the concentration-absorptivity product, is 4.05 X 10(-4) cm-1 for cobalt(II) in ethanol. Our preliminary detection sensitivity compares favorably with other laser-based spectrometric methods. This nonlinear laser technique is applicable to both fluorescing and nonfluorescing analytes.

Stable isotope ratio analysis at trace concentrations using degenerate four-wave mixing with a circularly polarized pulsed probe beam.

Stable isotope analysis based on vectorial optical-phase conjugation by resonant degenerate four-wave mixing (D4WM) is reported by using a D4WM method with vertically polarized pump beams and a circularly polarized probe beam. Since the polarization of the signal beam is different from that of the pump beams, the background radiation is suppressed more effectively. Excellent sensitivity, high spectral resolution, and efficient optical detection make this an effective and unusually convenient nonlinear spectrometric method for the analysis of trace amounts of stable isotopes. Using an excimer-pumped pulsed dye laser, the fine structures of lithium are examined. A detection limit of 2.5 ng/mL lithium is observed while a Doppler-free resolution is maintained by using transient "coherent-grating" based D4WM spectroscopy.

Laser analytical spectrometry based on optical phase conjugation by degenerate four-wave mixing in a flowing liquid analyte cell.

Nonlinear laser spectroscopy based on optical phase conjugation by degenerate four-wave mixing in an absorbing liquid analyte solution is reported as a sensitive analytical technique using a relatively low-power continuous-wave argon ion laser as the excitation source. This novel laser method provides excellent detection sensitivity since the analytical signal is a wavefront-reversed replica of the probe beam. Optical signal detection is convenient and efficient since the signal is a visible coherent laser beam. Important characteristics of this nonlinear laser method include cubic dependence of signal on laser power and quadratic dependence of signal on concentration. Excellent sensitivity, small detection volume, and convenient sample introduction offer many potential applications in trace-level condensed-phase analysis of continuously flowing systems. A preliminary detection limit of 2.9 X 10(-18) mol of eosin B in a simple flow cell is reported.

Stable-isotope ratio analysis based on atomic hyperfine structure and optogalvanic spectroscopy.

Atomic hyperfine structures were measured for the Cu I transition at 5782 A by optogalvanic spectroscopy at high resolution, with a cw dye laser. Samples were electro-deposited on the demountable cathode of a home-made hollow-cathode lamp. By spectral deconvolution, the relative isotopic abundances of (63)Cu and (65)Cu could be determined with good accuracy and precision. The technique is applicable to copper concentrations as low as 1.6 ppm.