Enhanced expression and clinical significance of CC-chemokine MIP-3 alpha in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most frequent visceral neoplasms worldwide. Using RT-PCR, ELISA, microdissection and immunohistochemistry, we investigated the expression profiles of CCL19, CCL20, CCL21 and CXCL12 and their receptors in tumourous and tumour neighbouring tissues from patients with HCC and in nonmalignant liver lesions, respectively. All chemokines were found to be expressed in normal liver and HCC tissues, yet CCL20 was the only chemokine showing significant upregulation in HCC tissues. Clinicopathological analysis revealed a distinct increase in CCL20 expression rates in HCC tissues of grade III tumours in comparison to HCC tissues from grade II tumours. On mRNA level, only chemokine receptor CCR6 revealed significant upregulation in HCC tissues. However, immunohistochemical studies indicated a marked CCR6 expression accumulated in a streak of normal cells along the tumour invasion front in all our HCC specimens which could provide a stimulative signal for the tumour to further expand. The present findings show significant overexpression of CCL20 in the tumour tissues and marked overexpression of the corresponding receptor CCR6 in the tumour invasion front of HCC patients in comparison to normal liver. Moreover, CCL20 expression was found to correlate with tumour grade and therefore, we suggest that the CCL20/CCR6 system may be involved in hepatocarcinogenesis.

Signal transduction by CXC chemokine receptor 4. Stromal cell-derived factor 1 stimulates prolonged protein kinase B and extracellular signal-regulated kinase 2 activation in T lymphocytes.

We report that stromal cell-derived factor (SDF)-1 has the remarkable capacity to induce sustained signaling through CXC chemokine receptor 4 (CXCR4). In contrast to other chemokines, such as monocyte chemotactic protein 1 (CC chemokine receptor 2 [CCR2]), macrophage inflammatory protein 1beta (CCR5), liver and activation-regulated chemokine (LARC [CCR6]), Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC [CCR7]), and IP10 (CXCR3), SDF-1 stimulates the prolonged activation of protein kinase B and extracellular signal-regulated kinase (ERK)-2. Activation of protein kinase B is reversed by displacement of SDF-1 from CXCR4 or inhibition of phosphatidylinositol 3-kinase. Although increasing concentrations of SDF-1 enhance CXCR4 internalization, kinase activation is prolonged. In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized. Furthermore, activation is prolonged by inhibiting SDF-1 degradation. The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.

A novel candidate oncogene, MCT-1, is involved in cell cycle progression.

Using the arbitrarily primed-PCR (AP-PCR) assay to detect genetic abnormalities that occur in a panel of lymphoid cell lines, we identified an amplified stretch of genomic DNA that contained a putative open reading frame. Northern blot analysis with this genomic clone revealed widespread low level expression in normal human tissue. The full cDNA sequence was obtained with no significant homology to any known genes in the genome database. We termed this novel gene with multiple copies in a T-cell malignancy as MCT-1. MCT-1 was localized to the long arm of chromosome Xq22-24 by flourescence in situ hybridization analysis. Although there was no significant homology at the primary sequence level, there was a limited degree of amino acid homology with a domain of cyclin H that appears to specify protein-protein complexes. This relationship between MCT-1 and cyclin H implied a potential role for MCT-1 in cell cycle regulation. Overexpression of MCT-1 increased the proliferative rate of cells by decreasing the length of the G1 phase without a reciprocal increase in the S and G2-M phases. Recent work has established the role of cell cycle regulatory molecules in the development of certain human malignancies. Therefore, we investigated the transforming ability of MCT-1 overexpression using soft agar growth assays and demonstrated that only MCT-1-overexpressing cells were able to establish colonies. Taken together, MCT-1 is a novel candidate oncogene with homology to a protein-protein binding domain of cyclin H.

G-Protein-coupled receptors and Fcgamma-receptors mediate activation of Akt/protein kinase B in human phagocytes.

Activation of the serine/threonine kinase Akt, also called protein kinase B (PKB), was investigated in human neutrophils. Stimulation of the cells with the chemoattractant fMet-Leu-Phe or the chemokines IL-8 and GROalpha leads to the rapid and transient activation of PKB. Maximum PKB activation correlates with the well documented kinetics of respiratory burst and exocytosis. Wortmannin, a selective inhibitor of phosphoinositide 3-kinases (PI 3-kinases) in neutrophils, abrogates PKB activation. Similarly homo and heterotypic cross-linking of FcgammaIIA and FcgammaIIIB causes a transient activation of PKB that is sensitive to wortmannin treatment. Kinase activity measurements in immunoprecipitates from lysates of the myelocytic GM-1 cells or GM-1/CXCR1 cells, which are transfected with the IL-8 receptor 1, confirmed the transient activation of PKB observed in neutrophils. Stimulation of human monocytes with the CC chemokine RANTES (regulated on activation normal T cell expressed and secreted) also results in the activation of PKB. Preincubation of monocytes and neutrophils with Bordetella pertussis toxin inhibits fMet-Leu-Phe and RANTES-stimulated PKB activation, demonstrating that coupling of the receptors to heterotrimeric Gi-protein is required. The data show, that activation of PKB by Gi-protein-coupled receptors is mediated by PI 3-kinase and suggest that PKB is a constituent of neutrophil activating pathways.

Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy.

Human prostate-specific antigen (PSA) has been widely used as a serum marker for cancer of the prostate. The cell type-specific expression of PSA also makes it a potential tumor antigen for prostate cancer immunotherapy. Study of the immunological aspects of PSA within either normal or malignant prostate tissue has been hampered by the lack of a mouse model, because no PSA counterpart has been identified in mice. Using a 14-kb genomic DNA region that encompasses the entire human PSA gene and adjacent flanking sequences, we generated a series of human PSA transgenic mice. In the six independent lines of transgenic mice generated, the expression of the human PSA transgene, driven by its own cis-acting regulatory elements, is specifically targeted to the prostate. Tissue distribution analysis demonstrated that PSA transgene expression closely follows the human expression pattern. Immunohistochemical analysis of the prostate tissue also showed that the expression of the PSA transgene is confined to the ductal epithelial cells. Despite expressing PSA as a self-antigen in the prostate, these transgenic mice were able to mount a cytotoxic immune response against PSA expressed by tumor cells, indicating that expression of the transgene has not resulted in complete nonresponsiveness. This transgenic mouse model will provide a well defined system to gain an insight into the mechanisms of nonresponsiveness to PSA, ultimately leading to strategies for immunotherapy of human prostate cancer using PSA as the target antigen.

Constitutive activation of protein kinase B and phosphorylation of p47phox by a membrane-targeted phosphoinositide 3-kinase.

BACKGROUND: Phosphoinositide 3-kinase (PI 3-kinase) activity is required for mitogenic signaling and for secretory responses. Cell activation is presumed to cause the translocation of PI 3-kinase from the cytosol to the plasma membrane where the kinase interacts with its substrate phosphatidylinositol (4,5)-bisphosphate. Thus, a membrane-targeted and therefore constitutively active kinase could help elucidate the role of PI 3-kinase in intracellular signaling. RESULTS: The membrane-targeting sequence of Ha-Ras, containing the consensus sequence for palmitoylation and farnesylation, was fused to the carboxyl terminus of p110 alpha, the catalytic subunit of PI 3-kinase. The lipid anchor directed PI 3-kinase to the membrane and led to constitutively elevated phosphatidylinositol (3,4,5)-trisphosphate levels in transfected cells. Expression of membrane-targeted PI 3-kinase resulted in the continuous activation of downstream effectors, such as protein kinase B (PKB, also known as Akt/RAC), which was recently shown to regulate glycogen synthase kinase-3. The constitutive activation of PKB was abolished by the specific PI 3-kinase inhibitor wortmannin, and PKB activation was marginal in transfectants expressing non-membrane-targeted PI 3-kinase. Multiple phosphorylation of the cytosolic factor p47phox is required for the rapid assembly of the phagocyte NADPH oxidase upon stimulation with agonists of G-protein-coupled receptors. We show here that the expression of membrane-targeted PI 3-kinase in the monoblastic cell line GM-1 results in a wortmannin-sensitive continuous phosphorylation of p47phox. CONCLUSIONS: Targeting of PI 3-kinase to the site of its preferred substrate leads to constitutive stimulus-independent enhanced catalysis and is sufficient to regulate different signal transduction pathways.

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes.

Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans.

Fundamental metabolic difference between cold stored lungs and livers and kidneys.

Short preservation times for lungs, are the main obstacle to exchange organs and increase the number of lung transplantations. In abdominal organ preservation UW solution has extended preservation times by including impermeable anions, adenosine and scavengers to prevent parenchymal cell swelling, to improve ATP regeneration and to inhibit reactive oxygen radicals. Metabolic changes in rabbit lungs, cold stored in UW-solution were compared with changes in rabbit livers and kidneys. Rabbit lungs swell during rewarming after a short storage period as opposed to livers and kidneys. They furthermore accumulate significantly more potassium while maintaining high ATP concentration throughout storage and reperfusion.

Changes in diurnal patterns related to changes in ozone levels.

Ozone is an ubiquitous air pollutant that affects both human health and vegetation. There is concern about the number of hours human populations in nonattainment areas in the United States are exposed to levels of O3 at which effects have been observed. As improvement in air quality is achieved, it is possible that O3 control strategies may produce distributions of 1-h O3 concentrations that result in different diurnal profiles that produce greater potential exposures to O3 at known effects levels for multiple hours of the day. These concerns have prompted new analysis of aerometric data. In this analysis, the change in the seasonally averaged diurnal pattern was investigated as changes in O3 levels occurred. For the data used in this analysis, 25 of the 36 sites that changed compliance status across years showed no statistically significant change in the shape of the average diurnal profile (averaged by O3 season). For 71 percent (10 out of 14) of the sites in southern California and Dallas-Fort Worth, Texas, that showed improvement in O3 levels (i.e., reductions in the number of exceedances over the years), but still remained in nonattainment, a statistically significant change in the shape of the seasonally averaged diurnal profile occurred. Based on the results obtained in this study, the evaluation of diurnal patterns may be useful for identifying the influence of changes in emission levels versus meteorological variation on attainment status. Using data from the southern California and Dallas-Fort Worth sites, which showed improvements in O3 levels, changes were observed in the seasonally averaged diurnal profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro metabolism of the antimalarial agent primaquine by mouse liver enzymes and identification of a methemoglobin-forming metabolite.

From a mouse liver microsomal system, we have isolated and identified a methemoglobin-forming metabolite of primaquine (PQ). Evidence has been found for both O-dealkylation and hydroxylation of PQ to form a metabolite, 5,6-dihydroxy-8-(4-amino-1-methylbutylamino)quinoline, which is highly active in forming methemoglobin in both normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes. It also actively decreases glutathione levels in glucose-6-phosphate dehydrogenase-deficient erythrocytes. The inhibitor SKF 525-A prevented metabolite formation while iproniazid and carbon monoxide did not inhibit metabolism completely but may have resulted in formation of a different unidentified metabolite. Mass spectrometry, HPLC, NMR, and other more indirect methods were used to help identify the metabolite. It was identified indirectly via a blue compound which results from extracting the actual metabolite from the incubation mixture with organic solvents under alkaline conditions in the presence of light. The blue compound was identified as a quinonimine in which the 8-amino side chain of PQ cyclizes to produce a third ring system.

Metabolism of 8-aminoquinoline antimalarial agents.

Some of the most effective antimalarial agents are derivatives of 8-aminoquinoline. The metabolic products of many of these compounds appear to be toxic to the erythrocytes of certain human subjects, especially those deficient in glucose-6-phosphate dehydrogenase. Although a number of studies have been conducted over many years, the metabolism of most of these compounds has not been determined. These studies are reviewed.Adult dogs dosed with tritium-labelled primaquine were observed to excrete approximately 16% of the injected radioactivity in the urine within 8 hours. Organic extracts of the urine were fractionated by thin-layer chromatography and the metabolic pattern obtained. Some primaquine was excreted along with at least five metabolites including 5-hydroxy-6-methoxy-8-(4-amino-1-methylbutylamino)quinoline (5HPQ) and a small amount of 6-hydroxy-8-(4-amino-1-methylbutylamino)quinoline (6HPQ). The 5HPQ could form a quinoneimine-type compound which may be a methaemoglobin-forming compound. This and other metabolites isolated from urine were found to be active methaemoglobin formers in in vitro studies. In vitro metabolism of primaquine by mouse liver enzymes also produced compounds capable of methaemoglobin formation. One of these had a blue colour when exposed to alkaline conditions, air, and light, and mass spectral data and nuclear magnetic resonance analysis indicated a structure similar to a 5,6-dihydroxy derivative of primaquine. However, the chemical structure of the metabolite was not identified in these studies.

Antiviral effect of cyclic amines on a 1969 isolate of A2 influenza.

Cyclooctylamine and amantadine inhibit the growth of 1969 isolates of A(2) influenza virus to a significant degree. There was slightly more inhibition of the virus by the cyclooctylamine (COA) than the amantadine; however, the dose of COA used was greater than the dose of amantadine. There was no significant difference between flasks treated 3 or 4 hr and those treated 2 hr. However, there was a curious relationship of more plaques in the flasks exposed to the two drugs for the longer intervals. Other experiments done with slight modifications in technique support the antiviral effect demonstrated in this experiment when the cell system is pretreated prior to virus infection. In two experiments, pretreating the cells for 2 hr with COA at 100 mug/ml but removing the drug solution and washing the cells prior to virus inoculation revealed no differences in plaque counts between controls and treated cells. This would indicate that the antiviral effect required the presence of the drug during the early stages of penetration of the cells by the virus particles.