Reactive oxygen-mediated damage to murine mammary tumor cells

We have shown, in a preliminary report, that macrophages can induce strand breaks in the DNA of co-cultured tumor cells (Chong et al., 1988). The present study is designed to determine if oxygen-centered species generated by the cell-free enzyme-substrate combination of hypoxanthine and xanthine oxidase can induce similar lesions and to identify the specific mediator(s). We report that co-incubation of murine mammary tumor cell lines with hypoxanthine and xanthine oxidase leads to the induction of DNA-strand breaks as determined by fluorescence analysis of DNA unwinding (FADU) assay or alkaline elution techniques. This damage is preventable by catalase which removes hydrogen peroxide but no protection is provided by agents to remove or prevent the formation of superoxide anion (superoxide dismutase), or hydroxyl radical (mannitol or the iron chelator o-phenanthroline). Likewise, cyclooxygenase or lipoxygenase inhibitors of arachidonate metabolism (indomethacin, nordihydroguaiaretic acid, caffeic acid) or bromophenacyl bromide do not alter the degree of DNA scission. Treatment with higher doses of oxygen species leads to significant toxicity as determined by evaluation of cell growth potential or colony-forming ability. Again, toxicity is prevented only by the presence of catalase. Tumor cells are able to rejoin strand breaks at lower, less toxic doses. When comparing different tumor cell subpopulations at various stages of progression, i.e., metastatic vs. nonmetastatic, for sensitivity to hydrogen peroxide-induced strand breakage, we found that at lower concentrations (less than 5μM) metastatic populations are sensitive whereas nonmetastatic populations exhibit no significant breakage. At higher concentrations of hydrogen peroxide, all lines were sensitive, suggesting that a lower threshold of sensitivity may exist for more progressed tumour cell lines.     

Immunotoxins up to the present day

Immunotoxins consist of monoclonal or polyclonal antibodies conjugated to bacterial or plant toxins. The toxins used are typically of the A-B type in which a toxic A chain is coupled to a B chain responsible for cell binding and facilitation of A chain entry into the cytosol. Two broad strategies have been followed: coupling intact toxins, or A chains alone, to antibodies. This review examines current progress inin vitro andin vivo research, including recent clinical studies, concentrating principally on ricin or ricin A chain conjugates. The future role of conjugates using membrane-acting toxins, immunolysins, is also discussed.     

Cell kinetic studies of endothelial cells in the adenocarcinoma EO 771 and the effect of cyclophosphamide

Cell kinetic studies of endothelial cells in the adenocarcinoma EO 771 growing in C57bl/6j mice and after transplantation into Balb/c-nu/nu mice, as well as of the effect of cyclophosphamide treatment have been carried out. The³H-thymidine labelling index of endothelial cells decreases from about 8% 3–6 days after tumour inoculation to about 3% at 18 days. This decrease parallels that of the labelling index of tumour cells, i.e. there is a positive correlation between the labelling index of endothelial cells and that of tumour cells. The labelling index of endothelial cells in the tumour periphery is two to three times as high as that in the tumour centre reflecting corresponding differences in the rate of proliferation. There is no difference in the proliferation of endothelial cells whether the tumour grows in C57bl/6j or in Balb/c-nu/nu mice. After treatment with cyclophosphamide the labelling index of endothelial cells decreases within 2 days to 1–2% and remains that low despite regrowth of the tumour with increased tumour cell proliferation, indicating that tumour relapse does not depend on tumour angiogenesis.     

Diagnosis of a peripheral neuroectodermal tumour by fine needle aspiration

One case of malignant peripheral neuroectodermal tumour successfully diagnosed by cytology is presented. Although a Papanicolaou stained smear could not lead to a diagnosis more specific than a malignant small cell tumour, ancillary analytic methods performed on the cytologic material including immunocytochemistry and electron microscopy yielded the correct diagnosis of peripheral neuroectodermal tumour. This case demonstrates that a precise categorization of small round cell tumours may be achieved by cytology as long as some material is kept for immunocytochemical and ultrastructural studies.     

Effects of tumour necrosis factor-α (cachectin) on glucose metabolism in the rat

Intravenous administration of a single dose (20 g) of recombinant tumour necrosis factor- (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-¹⁴C]glucose to ¹⁴CO₂ was significantly increased by the cytokine. In addition, [¹⁴C]lipid accumulation from [U-¹⁴C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of [³H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Ecological aspects of a silicified bivalve fauna from the Silurian of Gotland

The silicified Wenlockian (Silurian) bivalve fauna from MÖllbos, Gotland, is part of a life assemblage. The vast number of shells show unusual phenomena, e.g. shell repair, pearl and tumour formation, etc. A number of shells contain epibionts and bored, round holes. Presumptive predators of the bivalve community are discussed. Size-frequency distribution of the two most abundant species possibly reflects age classes. The fauna, comprising eleven species, is dominated by deposit-feeders (90 %). They exhibit niche diversification, including at least three different feeding levels within the sediment.     

担子菌多糖及羊脾RNA对肿瘤的抑制与有关组织中cAMP含量关系的探讨

本文讨论了对带瘤小鼠注射香菇多糖、松口蘑多糖和羊脾RNA后,活体内有关组织中cAMP含量与抑瘤率之间的关系。 结果表明,上述活性物质不仅增强了机体免疫活性,同时亦促使机体内脾、瘤等组织的cAMP增高,此种增高,与抑瘤率成正相关。     

Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Summary We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 by border and ca. 350 by internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration. The homology may promote close association of both termini of a T-DNA molecule on a target sequence; then TDNA may in some cases be integrated by a mechanism at least in part analogous to homologous recombination.Shogo Matsumoto is on leave from Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd, Ogaki, Gifu-ken 503, Japan     

Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography