BBA - Molecular Basis of Disease (v.1502, #2)

Pancreatic carcinoma is characterized by elevated content of hyaluronan and chondroitin sulfate with altered disaccharide composition by Achilleas D. Theocharis; Marina E. Tsara; Nikoletta Papageorgacopoulou; Dionisis D. Karavias; Dimitrios A. Theocharis (201-206).
The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.
Keywords: Pancreas; Carcinoma; Glycosaminoglycan; Proteoglycan;

A selective p38 MAP kinase (p38 MAPK) inhibitor, SB202190, induced apoptotic cell death of a macrophage-like cell line, J774.1, in the presence of lipopolysaccharide (LPS), as judged by DNA nicks revealed by terminal deoxy transferase (TdT)-mediated dUTP nick end labeling (TUNEL), activation of caspase-3, and subsequent release of lactate dehydrogenase. This cytotoxicity was dependent on both LPS and SB202190, and such inhibitors of the upstream LPS-signaling cascade as polymyxin B and TPCK blocked this macrophage cell death. SB202190 suppressed the kinase activity of p38, leading to inhibition of activation of MAPKAPK2 and then the subsequent phosphorylation of hsp27 in LPS-treated macrophages both in vitro and in vivo, but an inactive analog of SB202190, SB202474, did not. There was a threshold of the time of addition of SB202190 to LPS-treated macrophages to induce apoptosis, which was before full transmission of p38 activity to a direct downstream kinase, MAPKAPK2. Besides, localization of phosphorylated hsp27 in Golgi area of the LPS-treated macrophages was suppressed by SB202190, while it was not by SB202474. These results suggest that selective inhibition of p38 MAPK activity in LPS-induced MAP kinase cascade leads to apoptosis of macrophages.
Keywords: Apoptosis; Caspase-3; Erk; JNK; p38 MAP kinase phosphorylation;

Self-regulation of the endothelin receptor system in choriocarcinoma cells by Renate Mauschitz; Mila Cervar; Tom Hahn; Peter Pürstner; Gernot Desoye (224-234).
The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5–20 nM and 10 μM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.
Keywords: Endothelin-1; Receptor; Regulation; Secretion; Choriocarcinoma;

Protein–calorie malnutrition (PCM) represents a global health problem. The breakdown rate of glutathione S-transferase (GST) subunits determines their differential contents during protein depletion. Hepatic GST expression and the underlying mechanistic basis were investigated in PCM rats. PCM caused no change in rGSTA1/2 subunit. In contrast, rGSTA3/5 subunit was 2.4-fold induced during PCM, while the levels for rGSTM1 and M2 subunits were 30% and 70% suppressed. Increased GSTA3/5 expression was significantly prevented by cysteine or methionine treatment, although such treatment failed to restore the rGSTM2 level. In contrast to differential GST protein expression, PCM caused a 5–10-fold increase in rGSTA2/A3/A5 and M1 mRNAs, whereas rGSTM2 mRNA was 70% decreased. The elevations in rGSTA2/A3/A5 and M1 mRNAs were completely abolished by cysteine or methionine treatment during PCM, although the rGSTM2 mRNA level was not restored. PCM induced oxidative stress in the liver, as evidenced by protein carbonylation. Antioxidant response element (ARE)-binding activity of nuclear extracts from PCM rats was increased, which was immunodepleted with anti-Nrf-1/2 antibodies. Activation of nuclear ARE-binding proteins was inhibited by cysteine. Data showed that hepatic GSTs were differentially expressed during PCM, that certain GST mRNAs were increased with the ARE activation, and that cysteine was active in preventing increases in GST mRNAs and ARE activation.
Keywords: Protein–calorie malnutrition; Nrf-1/2; Cysteine; Methionine; Glutathione S-transferase; Antioxidant response element;

Huntington’s disease (HD) is caused by CAG repeat expansion in exon 1 of a large gene, IT15, possessing 67 exons. Transgenic mice expressing a truncated N-terminal peptide of huntingtin with an expanded polyglutamine tract translated only from exon 1 develop symptoms similar to Huntington’s disease. In the present study, a bacterial system (Escherichia coli) was used to express truncated peptides of huntingtin translated from exon 1 of the HD gene. Bacterial death was observed after the induction of peptides with expanded polyglutamine tracts, and both sodium dodecyl sulfate (SDS)-soluble peptides and insoluble aggregated material were detected by immunoblotting in the homogenates of such E. coli. E. coli death was partially reduced by the addition of dimethylsulfoxide (DMSO) or glycerol to the medium, with a consequent decrease in aggregated material and an increase in SDS-soluble peptide in the homogenate. These results suggest that DMSO and glycerol may decrease the toxicity of huntingtin with expanded polyglutamine tracts by acting as chemical chaperones.
Keywords: N-terminal huntingtin; Huntingtin; Huntingdon’s disease; Polyglutamine tracts;

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A2 by Anton J. Aarsman; Fred W. Neys; Hester A. van der Helm; Frans A. Kuypers; Henk van den Bosch (257-263).
During recent years, the high phospholipase A2 (PLA2) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA2. Recently the family of secreted low molecular mass PLA2 enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA2 during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA2 activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA2 activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA2 which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.
Keywords: Phospholipase A2; Serum; Acute chest syndrome; Trauma;

Pharmacokinetics of chronically administered all-trans-retinoyl-β-glucuronide in mice by Neil Sidell; Sayan Sawatsri; Michael J. Connor; Arun B. Barua; James A. Olson; Randal K. Wada (264-272).
After the subcutaneous injection of retinoyl β-glucuronide (RAG), both RAG and retinoic acid (RA), formed by the hydrolysis of RAG in vivo, achieved peak plasma concentrations within 1–2 h. Thereafter, RA was rapidly cleared from the plasma whereas RAG was eliminated much more slowly. No significant changes were noted in the peak (2 h) plasma levels of RAG for treatment periods up to 56 days (one injection of RAG/day), in the clearance rate of RAG from plasma, or in plasma retinol concentrations. Similarly, no consistent decrease in plasma levels of the RA hydrolysis product was observed. Mice undergoing these long-term chronic treatments with RAG did not show any clinical manifestations of retinoid toxicity. Taken together, our findings that chronic dosing with RAG produces sustained levels of both the parent compound and the RA hydrolysis product, combined with the apparent low toxicity of RAG, suggest that RAG could be a safe and useful alternative to some retinoids which are presently being utilized in the clinic.
Keywords: Retinoid; Retinoic acid; Retinoyl glucuronide; Pharmacokinetics; Mouse;

Vanadium (V) has recently been found to possess potent anti-neoplastic activity in rat hepatocarcinogenesis. Recent studies have suggested that the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], can inhibit growth and/or induce differentiation of a variety of cell types. In the present study, attempts have been made to investigate the combination effects on chromosomal aberrations (CAs) and DNA-strand breaks during the early preneoplastic stage of diethylnitrosamine (DEN)-induced rat liver carcinogenesis in male Sprague-Dawley rats. V (0.5 ppm ad libitum) and/or 1,25(OH)2D3 (0.3 μg/0.1 ml propylene glycol per os twice weekly) either alone or in combination were given to DEN-treated and control rats 4 weeks prior to DEN injection. Under these experimental conditions it was observed that, when given in combination, V and 1,25(OH)2D3 offered maximum protection against DEN-induced structural aberrations 96 h (66.7%, P<0.05), 15 days (44.9%, P<0.005) and 30 days (63.8%, P<0.001) after DEN injection. Synergistic supplementation of both V and 1,25(OH)2D3 4 weeks before DEN injection was found to offer significant (64.1%, P<0.001) protection against generation of single-strand breaks when compared with the DEN control. Thus, the combination effect of V, an essential trace element, and of 1,25(OH)2D3, a dietary micronutrient, appears beneficial in preventing genetic damage in liver cells upon alkylation induced by DEN.
Keywords: Vanadium; 1α,25-Dihydroxyvitamin D3; Chromosomal aberration; DNA-strand break;

The 18q21 region in colorectal and pancreatic cancer: independent loss of DCC and DPC4 expression by Victor M Barberá; Mercè Martı́n; Luisa Mariñoso; Assumpta Munné; Alfredo Carrato; Francisco X Real; Myriam Fabre (283-296).
The 18q21 region is frequently altered in gastrointestinal tumors. Three candidate tumor suppressor genes have been identified in it: DCC, Smad4/DPC4 and Smad2; the mechanisms involving their inactivation have not been completely elucidated. In this study, genetic losses at 18q21 and expression of DCC and DPC4 in colorectal (n=12) and pancreatic (n=16) cell lines and in colorectal tissues (n=10) were analyzed. The status of the 18q21 region was assessed using microsatellite analysis and duplex PCR of exonic sequences; expression was analyzed by RT-PCR; mutational analysis of DPC4 cDNA was performed in selected cases. Homozygous losses of microsatellite markers at 18q21 were not observed in colon or pancreas lines; however, a higher proportion of apparent homozygosity than expected was found. DCC and DPC4 transcripts were detected in 11/12 and 12/12 colorectal cancer lines, respectively. In tumors, homozygous losses at 18q21 were detected in three cases, without affecting DCC. All tumors retained DCC and DPC4 mRNA expression. In pancreatic lines, DPC4 was inactivated through homozygous deletion (n=5), intragenic mutation (n=3), and lack of protein (n=2). In conclusion: (1) microsatellite analysis does not provide adequate information regarding homozygous losses at 18q21; (2) approximately 65% of pancreas cancer lines show inactivation of DPC4; and (3) loss of DCC and DPC4 occur independently.
Keywords: Tumor suppressor gene; Microsatellite marker; Gastrointestinal cancer;

Bovine articular chondrocytes were maintained in high density pellet cultures with and without serum and nucleotide triphosphates for different periods of time. Despite half-lives in culture of about 3 h, adenosine triphosphate and uridine triphosphate in the presence of serum increased sulphated glycosaminoglycan and collagen deposition above control levels. In the presence of serum a single dose of uridine triphosphate on the first day of culture was sufficient to induce significant increases in subsequent proteoglycan and collagen deposition. We conclude that both adenine triphosphate and uridine triphosphate are anabolic for articular chondrocytes, and that this effect on the chondrocyte is long-term.
Keywords: Extracellular nucleotide triphosphate; Chondrocyte; Proteoglycan; Collagen;

Induction of collagenase-3 (MMP-13) in rheumatoid arthritis synovial fibroblasts by Bryan A. Moore; Sadie Aznavoorian; Jeffrey A. Engler; L.Jack Windsor (307-318).
There is a growing body of evidence that implicates matrix metalloproteinases (MMPs) as major players in numerous diseased conditions. The articular cartilage degradation that is characteristic of rheumatoid arthritis (RA) is believed to be mediated by the collagenase subfamily of matrix metalloproteinases. The preference of collagenase-3 (CL-3) for collagen type II makes it a likely candidate in the turnover of articular cartilage and a potential target for drug development. In this study, RA synovial membrane tissue was shown to express CL-3 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) and protein by immunohistochemistry. Fibroblasts isolated and cultured from RA synovial membrane tissue were induced to express CL-3 mRNA. CL-3 mRNA was detected after PMA treatment in 16 of the 18 RA synovial membrane fibroblast cell lines established for this study. These fibroblasts also expressed mRNA for collagenase-1 (CL-1, MMP-1), membrane type-1 matrix metalloproteinase, gelatinase A, gelatinase B, stromelysin-1, stromelysin-2, TIMP-1, and TIMP-2. They were further shown to express CL-1 mRNA constitutively and CL-3 mRNA only after stimulation with PMA, IL-1, TGF-β1, TNF-α, or IL-6 with IL-6sR. These fibroblasts also expressed after induction both CL-1 and CL-3 at the protein level as determined by Western blot analyses and immunofluorescence.
Keywords: Matrix metalloproteinase; Collagenase-3; Synovial fibroblast;