Paradoxical enhancement of interleukin-2-mediated cytotoxicity against K562 cells by addition of a low dose of methotrexate

Summary In vitro effects of methotrexate (MTX) on interleukin-2(IL-2)-mediated cytotoxicity of peripheral blood mononuclear cells (PBMC) were studied. PBMC were incubated with human recombinant IL-2 (25 U/ml) for 72 h; during the last 24 h, various concentrations (10 pM–1 µM) of MTX were added to the culture. Cytotoxicity against k562 cells was measured by a 4-h⁵¹Cr-release assay. The IL-2-mediated cytotoxicity was paradoxically increased at around a concentration (10 nM) MTX. Such a low concentration of MTX showed no anti-proliferative effect on cell growth. This enhancement with 10 nM MTX was shown only in an E-rosette⁺ (E⁺) population, but not in E-rosette^– (E^–). In addition, when E⁺ cells were treated with an anti-CD16 monoclonal antibody plus complement after incubation with IL-2 and MTX, MTX-induced enhancement was lost, suggesting that an E⁺CD16⁺ cell population was mainly involved in this augmentation. Positively sorted E⁺CD16⁺ cells showed similar enhancement of cytotoxicity after treatment with IL-2 plus MTX. On the other hand, MTX treatment did not show the phenotypical changes including of the E⁺CD16⁺ cells, indicating that this treatment did not affect the differentiation and proliferation of the specific cell subset. Our results indicate that a low dose of MTX could have a role in the regulation of immunological anti-cancer surveillance systems through the natural killer and lymphokine-activated cytotoxic cells.This work was supported in part by a Grant-in-Aid for Cancer Research (1–10) from the Ministry of Health and Welfare in Japan     

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.     

Ultrasonic assessment of pregnancy and fetal development in three species of macaque monkeys

Serial ultrasonic assessments of gestational sac (GS) and fetal biparietal diameter (BPD) were performed in the Japanese monkey (Macaca fuscata fuscata), rhesus monkey (Macaca mulatta), and crab-eating monkey (Macaca fascicularis). In the Japanese monkey, GS increased linearly for 3-8 weeks, whereas BPD increased in a linear-quadratic manner over 8 weeks to term. Ultrasonic assessments of spontaneously aborted fetuses with BPD growth retardation and diagnosis of a pelvic chocolate cyst also were reported.     

The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes

The genotoxicity of a variety of aniline derivatives was examined by a DNA repair test with rat hepatocytes. Out of 37 aniline derivatives, 6 chemicals, i.e., 2,4,6-trimethylaniline (mesidine), 2,4-xylidine, 3,5-diaminobenzoic acid, 3,4-diaminochlorobenzene, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline, elicited positive DNA repair responses. The results are in agreement with the bacterial mutagenicities with or without norharman of these compounds. Positive compounds of unknown carcinogenicity in the present assay, i.e., 3,5-diaminobenzoic acid, 2-chloro-4-methylaniline and 4-chloro-N-methylaniline are suspected of being potentially carcinogenic.     

Specific associations of fluorescent beta-2-microglobulin with cell surfaces. The affinity of different H-2 and HLA antigens for beta-2-microglobulin

We prepared single-labeled FITC derivatives of beta-2-microglobulin (b2m) and examined their interactions with class I MHC Ag H chains on living cells. Human b2m was reacted with FITC under mild conditions and separated by hydroxylapatite chromatography into three peaks containing single labeled derivatives of b2m peaks A, B, and C, and a peak containing the unmodified protein. The three fluorescent derivatives labeled the surfaces of cells bearing class I MHC Ag. The labeling was specific for class I MHC Ag as indicated by failure to label cells in the presence of excess unlabeled b2m and failure to label the HLA-negative cell lines Daudi and 721.221. Mouse cells labeled with fluorescent human b2m were recognized by mAb to the class I MHC Ag and by virus-restricted cytotoxic T lymphocytes, suggesting that labeling with the fluorescent b2m does not significantly alter the structure of class I MHC Ag or impair their ability to present viral antigens to cytotoxic T lymphocytes. We determined the kinetic and equilibrium binding parameters for the fluorescent b2m derivatives associating with the class I H chains of mouse and human cells. Peaks B and C exhibited biphasic binding to the mouse lymphoma cells EL-4(G-CSA-) (Kd1 = 1 x 10(-9) M; K2 = 1.5 to 3.0 x 10(-8) M whereas peak A bound to a small number of low affinity binding sites. In contrast to the biphasic binding observed with EL-4(G-CSA-), only monophasic binding was observed for peak C binding to RDM4 cells. Biphasic binding was also observed with the human B cell line LCL 721. Analysis of a series of LCL 721 class I MHC loss mutants and gene transferents revealed that the heterogeneity in binding is due to differences in the affinity of different class I encoded H chains for b2m.     

Preferential binding of E.coli histone-like protein HU alpha to negatively supercoiled DNA.

Binding specificity of histone-like HU alpha protein to supercoiled DNA was examined by gel retardation assay and chemical probing with OsO4. The latter method was proved to be a unique means for detecting torsional tension restrained in supercoiled plasmid in the presence of HU alpha. It was shown that HU alpha protein has preferential affinity to negatively supercoiled DNA relative to relaxed, nicked and linearized DNAs. There were two modes for binding of HU alpha to the supercoiled DNA: one was the binding associated with topological changes in DNA and the other was relatively strong binding, probably specific to certain particular structures of DNA. It was suggested that HU in vivo interacts preferentially with the regions deformed under torsional stress or with the metabolically active regions along DNA.     

Host range mutant of human immunodeficiency virus type 1: modification of cell tropism by a single point mutation at the neutralization epitope in the env gene.

We have isolated a variant of human immunodeficiency virus type 1 (HIV-1) which is highly infectious to fibroblastlike cells (BT cells) derived from human brain as well as CD4-positive T cells. This variant HIV-1, named HIV[GUN-1V], was obtained by infecting BT cells with a prototype HIV-1 isolate, named HIV[GUN-1WT], which is highly infectious to T cells but barely infectious to BT cells. HIV[GUN-1V] infects BT cells productively and this infection appeared to be mediated by CD4. To elucidate the viral gene responsible for the host range difference between the variant and prototype HIV-1s, we cloned and analyzed the provirus genomes of the two viruses. Examination of the infectivities of BT cells by various recombinant viruses and analyses of the nucleotide sequences of HIV[GUN-1V] and HIV[GUN-1WT] showed that a single nucleotide exchange was responsible for their difference in infectivity of BT cells: HIV[GUN-1V] contains a thymine residue instead of the cytosine residue in HIV[GUN-1WT] at position 931 of the env coding sequence. Replacement of cytosine by thymine at this position of the env coding sequence of the HIV[GUN-1WT] genome induced the ability to infect BT cells. The base exchange at this position was expected to change amino acid 311 of the envelope glycoprotein, gp120, from proline to serine, which is located in a variable region containing type-specific immunodominant epitopes. Thus, HIV[GUN-1V] acquired a wider host range than HIV[GUN-1WT] by a single point mutation in the env gene.     

Distribution of the pulmonary artery and vein of the orangutan lung

The distribution of the pulmonary artery and vein of the orangutan lung was examined. The right pulmonary artery runs obliquely across the ventral side of the right bronchus at the caudally to the right upper lobe bronchiole. It then runs across the dorsal side of the right middle lobe bronchiole. Thereafter it runs obliquely across the dorsal side of the right bronchus, and then along the dorso-medial side of the right bronchus. This course is different from that in other mammals. During its course, it gives off branches which run mainly along the dorsal or lateral side of each bronchiole. The left pulmonary artery runs across the dorsal side of the left middle lobe bronchiole, then along the dorso-lateral side of the left bronchus, giving off branches which run along each bronchiole. The pulmonary veins run mainly the ventral or medial side of, along or between the bronchioles. In the left lung, the left middle lobe vein has two trunks; one enters the left atrium, and the other enters the left lower lobe pulmonary venous trunk. This is also different from that found in most mammals. Finally, the pulmonary veins enter the left atrium with four large veins.     

The effect of platelet activating factor on ovulation.

The mechanism of ovulation has been compared to an inflammatory reaction. Platelet activating factor (PAF) is an important mediator of inflammation as it may induce the production of prostaglandins and lysosomal enzyme. We evaluated the potential role of PAF in PMSG-HCG induced ovulation using CV3988, a specific PAF receptor antagonist in a superovulated ICR mice (9-12 weeks old). CV3988 blocked the ovulation in a dose dependent manner, and the significant reduced ovulatory efficiency was observed at more than 500 micrograms dose (p less than 0.001). The ovulatory efficiency reduced by CV3988 was reversed by PAF in a dose dependent manner. In vitro fertilization (IVF) rate of follicular oocytes with treatment of CV3988 was not different from that of ovulated ova without treatment. These results suggest that PAF may be involved in the ovulation process but the presence of PAF may not be essential for the fertilization of the ova as IVF.