ABSTRACT
We investigated the early effects of low doses of ionizing radiation on the CD2 gene expression in normal human T lymphocytes in order to clarify if low-dose ionizing radiation has an enhancing and/or stimulatory effect on the immune response. The results indicate that even low doses of X-irradiation strongly enhance the appearance of CD2 antigen, both in PHA-stimulated and in resting T lymphocytes as demonstrated by a rosette assay or by immunofluorescence. Moreover, an accumulation of CD2 mRNA is observed in X-irradiated cells compared with non-irradiated, a fact that is attributed mainly to transcriptional activation of the CD2 gene and not to stabilization of preformed mRNA.
Subject(s)
CD2 Antigens/metabolism , T-Lymphocytes/radiation effects , CD2 Antigens/genetics , Cells, Cultured , Gene Expression Regulation/radiation effects , Humans , RNA, Messenger/metabolism , Radiation Dosage , T-Lymphocytes/immunology , Transcription, GeneticABSTRACT
Thioguanine resistant CHO cells (HPRT-) were stably cotransfected with pSV2-gpt and pi H3-CD2 vectors using the calcium phosphate coprecipitation technique. The effects of single low doses of ionizing radiation were studied in a CD2+ CHO clone. The CD2+ phenotype responsible for binding sheep erythrocytes and rosette formation, was not affected by X-rays doses in the range 2-6 cGy. However, after 10 cGy of X-irradiation, 50% of the cells lost the CD2+ phenotype. These results suggest that this CD2+ clone might be a very sensitive indicator of very low X-ray doses. The implications of the phenotypic changes, observed after very low doses of irradiation, are discussed.
Subject(s)
Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Surface/genetics , Antigens, Surface/radiation effects , Receptors, Immunologic/genetics , Animals , Antigens, CD/radiation effects , Antigens, Differentiation, T-Lymphocyte/radiation effects , CD2 Antigens , CHO Cells , Clone Cells/radiation effects , Cricetinae , Humans , Hypoxanthine Phosphoribosyltransferase , Phenotype , Radiation Dosage , Receptors, Immunologic/radiation effects , Rosette Formation , T-Lymphocytes/immunology , TransfectionABSTRACT
Interspecific cell hybrids between Chinese Hamster Ovary (CHO) and phytohaemagglutinin (PHA) stimulated human T lymphocytes were purified by preparative rosetting with sheep red blood cells (SRBC). The hybrid cell clone used in the present study consisted of cells containing a complete set of the 20 CHO chromosomes and one extra human chromosome, No 19. Hybrid cells constitutively expressed high levels of human CD2 surface receptor and formed multilayer rosettes with SRBC and human erythrocytes. In addition to CD2 they produced low levels of a small number of human extracellular proteins. These findings suggest that the factor(s) responsible for CD2 expression are produced by the hybrid and that genes responsible for CD2 expression are located on chromosome 19. However, the present work cannot exclude that material of chromosome 1, where the CD2 gene has been assigned previously, is integrated somewhere in the hybrid karyotype. Further work is needed to clarify this point.
Subject(s)
Antigens, Differentiation, T-Lymphocyte/genetics , Chromosomes, Human, Pair 19 , Receptors, Immunologic/genetics , T-Lymphocytes/immunology , Animals , Antigens, CD/genetics , Antigens, Differentiation, T-Lymphocyte/analysis , Antigens, Surface/genetics , Blotting, Southern , CD2 Antigens , CHO Cells , Clone Cells , Cricetinae , DNA/genetics , DNA Probes , Fluorescent Antibody Technique , Gene Expression , Humans , Hybrid Cells/immunology , Immunoblotting , Immunodiffusion , Karyotyping , Lymphocyte Activation , Phenotype , RNA/genetics , Receptors, Immunologic/analysis , Restriction Mapping , Rosette Formation , T-Lymphocytes/physiologyABSTRACT
We found that the formation of multilayer rosettes by transformed human blood lymphocytes after phytohemagglutinin (PHA) stimulation is correlated with conformational changes of the chromatin as seen by premature chromosome condensation (PCC). The frequency distribution of grades of PCC and multilayer rosette formation suggests that changes in chromatin are a prerequisite for rosette formation. Rosette formation was most pronounced for 24-h and 48-h cultures. Chromatin decondensation and rosette formation showed identical patterns. The possibility that multilayer rosette formation is directly dependent on conformational changes of chromatin is discussed.
ABSTRACT
Beta globulins, (Cohn Fr. III), are a major source of molecules affecting the DNA-synthesis of 3T3 and EAT cells. Growth inhibitors for both cell types, chromatograph at the same position, corresponding to a mol. wt of about 50,000. A very basic, (pI 10.1), factor is isolated by gel electrofocusing, which stimulates the DNA-synthesis of 3T3 and EAT cells. Because of its extremely high cationic charge and its adsorption on gels, the estimation of the exact molecular weight and its preparative isolation, becomes very difficult. Some of the above mentioned molecules are heat-stable and express their action even after boiling for 10 min at pH 3.
Subject(s)
Beta-Globulins/pharmacology , Carcinoma, Ehrlich Tumor/metabolism , DNA/biosynthesis , Animals , Beta-Globulins/isolation & purification , Cells, Cultured , DNA, Neoplasm/biosynthesis , Embryo, Mammalian , Growth Inhibitors/isolation & purification , Growth Substances/isolation & purification , Hot Temperature , Isoelectric Focusing , MiceABSTRACT
Evidence is presented to show the existence in human beta-globulin (Cohn fraction III) of 2 growth-promoting factors that stimulate DNA synthesis and cell division in human lymphocytes and 3T3 B mouse fibroblasts. After extraction of human beta-globulins at pH 3.0 in 0.1 M NaCl followed by sieve chromatography on Sephadex, 2 distinct fractions were obtained containing the biological activity; one of polypeptide nature and molecular weight of approx. 10 000 Daltons and the other consisting of a homogeneous ribonucleic acid, as revealed by polyacrylamide gel electrophoresis. The above-mentioned factors appear to lack both cell and species specificity, since they are mitogenic agents for cells as diverse as mouse fibroblasts and human lymphocytes.
Subject(s)
Beta-Globulins/pharmacology , Fibroblasts/cytology , Lymphocytes/cytology , Mitogens , Animals , Cell Division/drug effects , Cells, Cultured , Chromatography, Gel , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Hot Temperature , Humans , Lymphocytes/drug effects , Mice , Stimulation, ChemicalABSTRACT
A general equation is derived describing data of DNA-RNA hybridization in the presence of a competing self-annealing reaction of RNA. The well known double-reciprocal relation and the Scatchard equation are shown to be limiting cases of this general equation. Some new hybridization data at various temperatures are presented and analysed by using the new equation. The results can only be explained if we assume that the behavior of DNA towards single RNA molecules is the same as that towards the annealed form, (RNA12. The variation of the equilibrium constant of the hybridization reaction with temperature is small, indicating a small heat of reaction. The maximum amount of hybridized RNA at equilibrium appears to be independent of temperature.
