Initiating activity of quinones in the two-stage transformation of BALB/3T3 cells

2-tert-Butyl-1,4-benzoquinone (BQ), a metabolite of the rodent carcinogen 3-tert-butyl-4-hydroxyanisole (3-BHA), has been shown previously to have initiating activity for cell transformation. In this paper, we examined the initiating activity of quinones in a two-stage transformation assay using BALB/3T3 cells. Cells were treated first with a quinone and then with the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA). The quinones tested were 1,4-benzoquinone (pQ), phenyl-1,4-benzoquinone (PhQ), menadione and 2,5-di-tert-butyl-1,4-benzoquinone (DBQ) in addition to BQ. pQ is a metabolite of benzene and phenacetin, and PhQ is a metabolite of o-phenylphenol (OPP) and sodium o-phenylphenate (OPP-Na). All of the tested quinones induced transformation in the presence of TPA but not in its absence. The extent of transformation caused by quinones followed by TPA was weak but statistically significant. Thus these quinones were shown to act as initiators in the transformation of BALB/3T3 cells. This result suggests that BQ, pQ and PhQ may be involved in carcinogenesis by 3-BHA, benzene, phenacetin, OPP and OPP-Na in vivo. Menadione has been reported to cause cytotoxic effects and mutations through active oxygen generation from semiquinone radicals. DBQ has two bulky substitutes which interfere with covalent bonds with DNA. Menadione and DBQ exhibited initiating activity in the present study. This result suggests that active oxygens generated from semiquinone radicals may play a role in the initiation of cell transformation.     

Comparison of propylene oxide and epichlorohydrin effects in two transformation tests (C3H/10T1/2 and SHE cells)

The neoplastic cell transformation induced by propylene oxide (PO) and epichlorohydrin (ECH) was studied in two in vitro assays, mouse embryo fibroblasts (C3H/10T1/2) and Syrian hamster embryo (SHE) cells. In C3H/10T1/2 cells treated with PO (2.5-10 mM), the transformation frequencies were enhanced about 2-4 times in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), compared with the transformation frequencies in the absence of TPA. In SHE cells, an even higher increase (about 6-9 times) was reached at concentrations of 2.5-20 mM. The presence of TPA strongly influenced the ability of ECH to induce the morphological transformation at low-moderate concentrations (0.25-1 mM). At the highest concentrations applied, 1 mM in C3H/10T1/2 cells and 0.5 mM in SHE cells, 41- and 4-fold increases, respectively, were observed. In C3H/10T1/2 cells, the rad-equivalence (rad/mMh) of PO and ECH in the presence of TPA was calculated to be 36 +/- 8 and 296 +/- 65 (mean +/- S.E.), respectively.     

GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1)

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AA and PEt formation remained unchanged. In conclusion, in alphaT3-1 cells the GnRH-induced homologous desensitization affects the GnRH coupling with PLC, PLA2 and PLD by mechanism(s) which do not implicate TPA-sensitive PKC isoforms, but likely reflect time-dependent modification(s) on the activation processes of the enzymes.     

Acute transformation of chronic myelomonocytic leukemia with t(1;3) (p36;q21) abnormality

A 66-year-old man was given a peripheral blood test because of low grade fever. Leukocytosis was detected, and the blood and bone marrow findings were consistent with those of chronic myelomonocytic leukemia. Three months later the hematological findings were: WBC 58,800/microliter (19% blastoid cells, 22% monocytes), Hb 9.0 g/dl, and a platelet count of 116 x 10(4)/microliters. A bone marrow examination revealed the presence of 52.6% blastoid cells and dysmegakaryocytopoiesis, including micromegakaryocytes. Serum and urinary lysozyme levels were elevated. Karyotypic analysis detected t(1; 3) (p36;q21), but not major bcr/abl mRNA. The patient was given a diagnosis of acute transformation of chronic myelomonocytic leukemia. Despite treatment, he died about 3 months later. t(1;3) is occasionally observed in cases of myelodysplastic syndrome (MDS) and leukemia. Patients with t(1;3) often exhibit dysmegakaryocytopoiesis; furthermore, acute leukemia develops more readily in those who also have MDS. Cases of long-term survival are rare.     

Hemocompatibility studies of surface-treated polyurethane-based chronic indwelling catheters

The objectives of this research were to evaluate and compare the interactions of several polyurethane-based central venous catheter materials with blood. Specifically, measurements of fibrinogen adsorption, platelet adhesion, kallikrein generation, and fibrinopeptide A (FPA) release were performed. The catheter materials examined in this study included: platinum-cured, 50 shore A durometer, barium sulfate-filled, silicone (SI); Tecoflex EG85A-B20 polyurethane (PU); PU catheters whose outer surface had been impregnated with ion beam-deposited silver atoms (AgI and AgII); PU catheters coated with a hydrophilic, polyacrylic acid polymer (UC); PU catheters coated with an air-cured PTFE emulsion (CS); and PU catheters coated with an aminofunctional dimethylsiloxane copolymer (JG). The time course of fibrinogen adsorption from plasma to the SI, JG, PU, and CS materials was similar, with CS exhibiting the least amount of adsorbed fibrinogen after 1 h (65 +/- 4.7 ng cm-2) and PU the greatest (144 +/- 16.5 ng cm-2). After 90 min of contact, AgI and AgII exhibited the greatest number of adherent platelets, levels that were approximately two to three times higher than those on the other catheter materials. With the exception of UC and PU, which caused kallikrein generation levels approximately half that of the positive (glass) control, little kallikrein formation was observed for any of the materials relative to the negative control. Finally, FPA generation was greatest using the SI, CS, and PU materials, with the latter causing the production of almost four times the amount of FPA as the negative control. This preliminary assessment of the hemocompatibility of the various catheters suggests that the surface treatments did not adversely affect their interactions with blood components; further investigations of these materials are therefore warranted in order to completely characterize their behavior prior to use in clinical situations.     

Molecular characterization of a variant Ph1 translocation t(9;22;11) (q34;q11;q13) in chronic myelogenous leukemia (CML) reveals the translocation of the 3'-part of BCR gene to the chromosome band 11q13

We performed cloning and sequence analysis of translocation junctions at 11q- and 22q- (Ph1) chromosomes and the corresponding germline DNAs of a variant Ph1-positive CML with t(9;22;11)(q34;q11;q13). Southern blot analysis using probes for different regions of bcr mapped the translocation break near the 5'-side of bcr exon 4. Cloning, Southern blot analysis and restriction map analysis of both bcr fragments showed that the part of bcr 3'- to the translocation break moved to 11q13. Sequence analysis of the translocation junction on the Ph1 chromosome showed that the translocation break occurred 63 bp upstream of exon 4. Compared to the germline sequence, bcr sequence from the translocated partners showed deletion of seven basepairs at the site of translocation. A probe derived from the 5'-region of the clone isolated from the 11q- chromosome identified clonal rearrangements in the leukemic DNA. Restriction map and sequence analysis showed that this clone consisted of the 3'-half of the glutathione S-transferase Pi (GST-Pi) gene and the 3'-part of bcr. We identified two point mutations in the GST-Pi allele involved in translocation. Northern blot analysis showed that the GST-Pi gene was expressed in the leukemic cells at blast crisis but not at chronic phase; however, no fusion mRNA between GST-Pi and bcr was identified. We did not find any sequence homology between 11q13 DNA and 22q11 DNA around the translocation breakpoints; however, sequences homologous to ALU repeats were identified close to the sites of translocation breaks at 22q11 and 11q13. This study supports our hypothesis that variant Ph1 translocations may occur as primary cytogenetic changes similar to the classical Ph1 translocations.     

Circadian clock controlling arylalkylamine N-acetyltransferase-like activity in the cricket (Gryllus bimaculatus) egg

Insulin suppresses hepatitis B surface antigen (HBsAg) gene expression and stimulates cell proliferation in human hepatoma Hep3B cells. 12-O-tetradecanoyl phorbol-13-acetate, TPA, has been demonstrated to mimic insulin actions in these cells. We examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the signaling pathways of insulin and TPA towards these two biological phenomena in Hep3B cells. The pre-treatment of 5 microM of wortmannin diminished insulin suppressed HBsAg production and completely abolished insulin stimulated cell proliferation. However, wortmannin had no effect on TPA actions in both HBsAg suppression and cell growth stimulation. We further investigated the effect of wortmannin in mitogen-activated protein kinases (MAPKs) activation induced by insulin or TPA. After the pretreatment of wortmannin, insulin activated MAPKs was completely blocked, but TPA was still capable to activate MAPKs. These results suggest that PI 3-kinase is involved in insulin actions but not in TPA effects, and allow us to dissociate the signaling pathways of insulin and TPA in human hepatoma Hep3B cells.     

Application of enzyme immunoassays for testing haemocompatibility of biomedical polymers

In this study enzyme immunoassays are presented for the assessment of platelet adhesion/activation and fibrinogen adsorption/conformation. The estimation of platelet adhesion and activation was performed with two enzyme immunoassays (EIAs) using monoclonal antibodies directed against CD42b (GP lb) and CD 62 (GMP 140 or P-Selectin). The applicability of EIA was first demonstrated in microtitre plates coated with fibrinogen. The thrombogenic substrate showed that platelet adhesion and activation reached a plateau level within 30 min. The use of EIA for testing biomaterials was demonstrated with polymeric reference materials where a differentiation of materials with respect to adhesion and activation was achieved. To validate the EIA scanning electron microscopy was applied and confirmed the different extent of adhesion and activation of platelets on reference materials. In addition, polyurethaneureas, based on 4,4'-diphenylmethane diisocyanate and polytetramethylene glycols, with different hard segment content and composition were investigated. It was found that both adhesion and activation were not simply dependent on the hard segment content but also on the hard segment composition. To get more insight into the mechanism of this process, two EIAs for the binding of fibrinogen using polyclonal and monoclonal antibodies were developed. There it was shown that the adhesion and activation of platelets on polyurethaneureas was not simply dependent on the total amount of adsorbed fibrinogen but rather on its conformation, indicated by the binding of the monoclonal antibody directed vs the gamma-chain of fibrinogen.     

Influence of chronic variable stress (CVS) on the association of glucocorticoid receptor with heat-shock protein (HSP) 90 in rat hippocampus

Brevetoxins are produced by the marine dinoflagellate Ptychodiscus brevis, an organism linked to red tide outbreaks, and the accompanying toxicity to marine animals and to neurotoxic shellfish poisoning in humans. Brevetoxins bind with high affinity to voltage-sensitive sodium channels and cause increased sodium ion conductance and nerve cell depolarization. The brevetoxin competitive binding assay with tritium-labeled brevetoxin 3 (3H-PbTx-3) and rat brain synaptosomes is a sensitive and specific assay for pure brevetoxins. Here we report that extracts of manatee, turtle, fish, and clam tissues contain components that interfere with the assay by cooperative, noncompetitive inhibition of 3H-PbTx-3 specific binding and increased nonspecific binding to synaptosomes. By determining the "apparent" toxin concentration ("[Toxin]") in the extract at several assay concentrations, a reasonable correction for the complex inhibition could be made using a semilog plot to extrapolate [Toxin] to zero extract concentration to obtain [Toxin]0. Spiking 4 extracts with 60 nM PbTx-3 caused [Toxin]0 to increase by 41 +/- 8 nM, indicating that the noncompetitive components did not prevent the assay of toxin but did reduce the accuracy of the result. Fourfold repetition of the assay of 4 samples gave standard deviations of 25 to 60% of the value of [Toxin]0, so the error can be fairly large, especially for samples with little toxin. Purification of an extract with a 1 g sample prep column of C-18 decreased the complex inhibition by about 3-fold but did not eliminate interference in the assay.     

Comparison of tissue polypeptide antigen (TPA) with cancer antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) in follow-up of breast cancer

Cancer antigen 15-3 (CA 15-3), carcinoembryonic antigen (CEA) and tissue polypeptide antigen (TPA) were measured in 679 sera of breast cancer patients and in 94 sera of women without breast cancer. The tumour markers were determined using immunoluminometric assays (ILMA). The assays are characterised by an inter-assay-imprecision and intra-assay-imprecision <4 %. The breast cancer patients were staged according to the TNM classification stage 0-IV (by UICC) in patient groups with a compatible prognosis. Median and range of each stage were investigated. The cut-off values (95th and 97.5th percentile of control group) of CA 15-3, CEA and TPA were determined; specificity, sensitivity, positive and negative predictive value (PV) and efficiency were investigated for these cut-off's and the receiver operating characteristic (ROC) curves were calculated. The differences between control group and stage 0-3 were shown as non-significant for CA 15-3 and CEA but significant for TPA. Significant differences were found in stage 4 for all three tumour markers. The three tumour markers did not have differences in specificity, positive and negative PV and efficiency. TPA and CA 15-3 demonstrated comparable results in sensitivity and ROC curve analyses. These results were better than those from CEA.     

Structural aberrations of the long arm of chromosome no. 22. Report fo a family with translocation t(11;22) (q25;q11)

A chromosomal translocation t(11;22) (q25q11) is described in a family. Four members, in two generations, had the same translocation but showed phenotypic variation. Case reports of chromosome aberrations involving the long arm of chromosome 22 associated with and without chronic myeloid leukemia (CML) are reviewed. It appears that the distal segment of the long arm or chromosome 22 is either translocated or deleted, resulting in congenital anomalies, presumably due to chromosome imbalance. In other instances, a specific breakpoint on 22q results in the origin of Philadelphia chromosome (Ph1) associated with CML.     

Cell-mediated cytotoxicity for melanoma tumor cells: detection by a (3H) proline release assay

An in vitro lymphocyte-mediated cytotoxicity assay using [3H]proline-labeled target cells is described. The assay, modified from an original procedure of Bean et al., assesses the release of [3-H]proline by filtering the total culture fluid containing both trypsinized tumor cells and effector cells. Filtration is performed with a semiautomatic harvesting device using low suction pressure and large-diameter glass filters. Pretreatment of filters with whole serum diminishes adsorption of cell-free radioactive material considerably and thus increases the sensitivity of the assay. Nearly 100% of the radioactivity could be recovered with this harvesting device. The technique allowed the detection of cytolytic activities of lymphocytes after 6 h of incubation. Lymphocytes from patients with primary malignant melanoma showed a significantly higher cytolytic reactivity (P less than 0.001) than normal donors' lymphocytes against three different melanoma cell lines. In a series of parallel experiments on 36 patients and 18 normal donors, this modification of the [3H]proline test was compared with three different assays: the conventional microcytotoxicity test of Takasugi and Klein, the original [3H]proline microcytotoxicity test of Bean et al., and the viability count of tumor cells.