The effects of expression of human c-myc and both mutated (T24) and normal forms of human Ha-ras-1 were studied in an aneuploid rat fibroblast line (208F). Mutated T24 Ha-ras was also studied in a near-diploid cell derived from early passage Chinese hamster lung fibroblasts (CHL). In contrast to the parental fibroblasts, cells expressing any of the human oncogenes engendered rapidly growing tumours in immune-suppressed animals. Blood- and lymph-borne metastases were observed from both ras- and myc-expressing cells. In general ras-expressing cells were more aggressive than those expressing myc. In the 208F background, expression of c-myc was associated with an incidence of mitosis similar to that in tumours expressing T24 Ha-ras, but incidence of single cell death by apoptosis was higher. Quantitatively, expression of human oncogene mRNA was constant during growth in vivo, and similar to that sometimes observed in human neoplasms. Of 9 endogenous proto-oncogenes, 7 showed no change in expression from the parental fibroblasts, but c-abl and c-fos were strongly expressed in all cells expressing human ras or myc. Thus these tumorigenic cells, although transfected with single human oncogenes, all expressed oncogenes with both nuclear- and membrane-associated products.

Animals↗

PubMed1986

The Edinburgh intercalated honours BSc in pathology: evaluation of selection methods, undergraduate performance, and postgraduate career.

In a study of 60 students who entered the intercalated honours BSc course in pathology at the University of Edinburgh over 10 years the conventional criteria of academic excellence and motivation were shown to be appropriate for the selection of honours students. When compared with classmates who did not take the intercalated year but who had shown similar high academic ability in the preclinical course the students who had taken the honours BSc did better in the remainder of the undergraduate curriculum. Of 42 honours students, 18 (43%) entered academic careers, particularly in pathology and medicine, but there was no observed tendency for students without honours BSc to do so. Although it is impossible to establish a causal relation between taking the honours course and subsequent academic distinction, the results suggest that the intercalated honours BSc in pathology serves a useful function in introducing able students into academic careers. The findings justify the financial support made available to such students during their intercalated year by the Medical Research Council and the Scottish Education Department.

Achievement↗

PubMed1986

Evaluation of a monoclonal antibody to ras peptide, RAP-5, claimed to bind preferentially to cells of infiltrating carcinomas.

RAP-5, a monoclonal antibody raised against a p21ras peptide, has been claimed to show immunohistochemical localisation of cells with infiltrative properties in human tumours. We confirmed that this antibody reveals pronounced cellular heterogeneity in human colonic neoplasms but could find no obvious relationship to infiltrative activity. RAP-5 bound to many different cell types, neoplastic and normal. In order to clarify the specificities of RAP-5 we applied it to two cell lines: nontumorigenic hamster fibroblasts in which ras expression is barely detectable, and a vigorously tumorigenic line derived from these fibroblasts by insertion of the human mutated Ha-ras oncogene in a high expression vector. Another antibody to p21ras, Y13-259, clearly distinguished between these cell lines both on immunoblots and immunocytochemically, but RAP-5 did not. Rather, it bound to proteins of a variety of molecular weights in both cell lines. The results show that RAP-5 is unlikely to be a useful reagent for detection of ras associated proteins in human tissues.

Adenocarcinoma↗

PubMed1986

What is apoptosis?

Explore the source record for details and available documents.

Cell Nucleus↗

PubMed1985

Enhancement of DNA-mediated gene transfer by high-Mr carrier DNA in synchronized CV-1 cells.

Synchronized CV-1 cells were transfected with SV40 (simian virus 40) DNA-calcium phosphate co-precipitates. In the presence of carrier DNA, the transfection efficiency of SV40 DNA was decreased 5-fold in S-phase cells and was increased 4-fold in preparations of mitotically enriched cells as compared with asynchronous controls. No difference was observed when carrier DNA was omitted, when cells had progressed through S-phase and into G2-phase, or when the infectivity of cells to intact SV40 virus was tested. These results highlight the importance of cell-cycle-dependent factors on DNA-mediated gene transfer.

Animals↗

PubMed1985

Immunohistochemical detection of the ras oncogene p21 product in an experimental tumour and in human colorectal neoplasms.

The monoclonal antibody Y13 259 to the ras oncogene protein product p21 was used in an immunohistochemical study of ras expression in human colorectal neoplasms. The ability of the antibody to detect enhanced levels of ras expression was confirmed by its use with an experimental neoplasm known to express ras at high levels. Human colonic adenocarcinomas in general showed a similar staining intensity to that seen in normal mucosa. Adenomas however showed consistently high p21 expression as indicated by staining intensity. This suggests that elevated ras expression may be important in the development of adenomas, but that high levels need not be sustained in the conversion to invasive carcinoma.

Adenocarcinoma↗

PubMed1985

A cellular abnormality in glucocorticoid resistant asthma.

Peripheral blood mononuclear cells from asthmatic patients were cultured in soft agar with or without the synthetic glucocorticoid methylprednisolone (MP). The resulting colonies consisted mostly of T lymphocytes but included monocytes, often in a central position. Colony growth from patients whose asthma had responded satisfactorily to glucocorticoid medication was inhibited by MP in vitro at concentrations as low as 10(-9) M, and the ratio of helper to suppressor/cytotoxic T cells was reduced relative to untreated cultures. In contrast, patients shown to be resistant to the therapeutic effects of glucocorticoids yield colonies which differed little in number, size or constituent phenotype, whether untreated or exposed to MP at concentrations as high as 10(-8) M. Higher concentrations inhibited colony growth from both types of patient. Hybrid colonies were generated from partially purified cells from pairs of patients: monocytes from one member and lymphocytes from the other. The steroid sensitivity of these colonies was found to be dependent upon the source of the monocytes and not the lymphocytes. The results support the view that, in glucocorticoid resistant asthma, there is a defect in monocyte responsiveness to the hormone. This defect could explain the poor therapeutic effect of glucocorticoids in these patients. The results suggest that monocyte factors may be of importance in the pathogenesis of asthma in general.

Asthma↗

PubMed1985

Macrophage recognition of cells undergoing programmed cell death (apoptosis).

As a model for the recognition of effete cells by their viable neighbours. BALB/c mouse thymocytes were coincubated with isologous peritoneal macrophages. The macrophages bound preferentially to thymocytes undergoing apoptosis, a mode of death induced in these cells by treatment with the glucocorticoid hormone methyl-prednisolone. Binding occurred in the absence of serum and was inhibited by N,N'-diacetyl chitobiose, N-acetyl glucosamine and, to a lesser extent, by N-acetyl galactosamine and D-galactose. L-fucose, D-mannose and N-acetyl neuraminic acid had no effect. The results suggest the presence of lectin-like molecules on the surface of the macrophage that recognize changes in the cell-surface carbohydrate of the apoptotic cell. The pattern of inhibition of binding by monosaccharides differs from that of previously described endogenous mammalian lectins.

Animals↗

PubMed1984

The uptake and stability of simian-virus-40 DNA after calcium phosphate transfection of CV-1 cells.

The uptake and fate of purified SV40 (Simian virus 40) DNA, transfected into permissive CV-1 cells by calcium phosphate precipitates, was examined. By using a viral plaque assay, optimum conditions for transfection were established and transfection efficiencies of up to 10(6) plaque-forming units/micrograms of SV40 DNA were obtained. After a 2h exposure to 3H-labelled SV40 DNA-calcium phosphate co-precipitates under basal conditions, up to 7% of the input DNA became cell-associated, with approx. 4% reaching the nuclear fraction. This value was never exceeded, even under conditions known to enhance significantly the ultimate transfection efficiency, such as increased exposure time, addition of carrier DNA or treatment with DMSO (dimethyl sulphoxide) or glycerol. Substantial degradation of this SV40 DNA occurred within a further 4h, apparently in both nucleus and cytoplasm. Degradation of form-II and form-III SV40 DNA, which have lower transfection efficiencies than form-I DNA, was no more rapid than degradation of form-I DNA. The results indicate that less than 0.5% of the transfected DNA which reached the nucleus is protected from nuclease attack. The mechanism of action of agents such as glycerol, DMSO or carrier DNA remains obscure, but they may be involved in conferring greater stability to the intracellular SV40 DNA rather than merely affecting its rate of entry into the cell.

Animals↗

PubMed1984

Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis.

In glucocorticoid-treated rat thymocytes and the murine lymphoid cell lines L5178 and S49 the morphology of apoptosis is associated with chromatin cleavage. The cleavage is at internucleosomal sites, apparently through activation of an endogenous endonuclease. In variants of the cell lines selected for resistance to glucocorticoid, neither apoptosis nor chromatin cleavage were observed after steroid treatment, and steroid receptors were undetectable. In thymocytes, both the morphological changes of apoptosis and chromatin cleavage were inhibited by cycloheximide and actinomycin D. The calcium-magnesium ionophore A23187 induced apoptosis and chromatin cleavage in thymocytes, and these effects were also inhibited by cycloheximide. The data confirm that the condensed chromatin which characterizes apoptosis morphologically consists of endogenously digested chromatin fragments. They also provide support for the view that at least some cells enter apoptosis by a process dependent upon macromolecular synthesis.

Animals↗

PubMed1984

Resistance to methylprednisolone in cultures of blood mononuclear cells from glucocorticoid-resistant asthmatic patients.

In order to investigate the cellular mechanism of glucocorticoid resistance in chronic asthma, peripheral blood mononuclear cells (MNC) from asthmatic patients were cultured in soft agar. Cells from patients known to be clinically sensitive to glucocorticoid therapy did not differ significantly from those of clinically resistant patients in terms of their immunophenotype or the number of colonies generated by culture in the presence of phytohaemagglutinin. The glucocorticoid methylprednisolone (MP) at low concentration (10 nmol/l) inhibited colony growth from cells of glucocorticoid-sensitive patients, whereas there was much less inhibition of colony growth from resistant patients' cells. In a small prospective study inhibition of colony growth by methylprednisolone in vitro correlated with the subsequently determined sensitivity of the patients' asthma to glucocorticoid therapy. Assessment in vitro of glucocorticoid sensitivity may help to predict which patients may be spared ineffectual glucocorticoid medication. The results raise the possibility that peripheral blood mononuclear cells may respond to glucocorticoid in a similar manner to cells involved in the pathogenesis of asthma.

Asthma↗

PubMed1984

Hormone-induced cell death. 2. Surface changes in thymocytes undergoing apoptosis.

In vivo, apoptotic cells are swiftly recognized by phagocytes, presumably because of changes on their surface. This article describes surface changes in rat cortical thymocytes undergoing apoptosis induced by glucocorticoid treatment in vitro. Homogeneous populations of thymocytes early in apoptosis were prepared by isopyknic centrifugation. These cells were compared with purified nonapoptotic cells in terms of several surface characteristics, including binding to macrophages, surface ultrastructure, microelectrophoretic mobility (a measure of surface charge density), and ability to bind four lectins and four monoclonal antibodies to thymocyte antigens. Apoptotic cells bound to macrophages more avidly than did nonapoptotic cells by a process not dependent upon serum factors. Their surfaces lost microvilli and became " blistered ," apparently through fusion of vesicles of endoplasmic reticulum with the plasma membrane. The surface charge density of apoptotic cells was less than that of nonapoptotic cells. Surface antigens and lectin-binding sites were less abundant on apoptotic than on normal cells, in proportion to the general reduction in cell size observed in apoptosis. Differences between apoptotic and normal cells were not detected, however, in the relative quantities of exposed galactose, N-acetyl galactosamine, N-acetyl glucosamine, N-acetyl neuraminic acid, or of several surface antigens, including the major sialoglycoproteins of the thymocyte membrane. It appears that although several changes occur in the surface of apoptotic cells, many cell membrane structures remain intact. The changes responsible for the recognition of apoptotic cells by phagocytes are more subtle than those detectable by the binding of lectin and antibody probes, but preliminary data suggest that a lectin-sugar interaction is involved.

Animals↗

PubMed1984

The mode of cell death associated with cavitation in teratocarcinoma-derived embryoid bodies.

Cell death occurring in embryoid bodies derived from the embryonal carcinoma cell line, PSA4, which undergo cavitation, and in those from the related cell line S2, which do not undergo cavitation, was classified as apoptosis or necrosis by ultrastructural criteria. Both modes of cell death were seen in PSA4 embryoid bodies while apoptosis alone was seen in S2 embryoid bodies. No significant difference was found between PSA4 and S2 embryoid bodies either in apoptotic incidence score or in the spatial distribution of apoptotic events. We therefore conclude that although apoptosis and tissue modelling coexist in PSA4 embryoid bodies, necrosis rather than apoptosis is causally related to formation of the cavity.

Animals↗

PubMed1983

Hyperthyroidism due to a thyrotrophin-secreting microadenoma.

We report a 63-year-old female with a TSH-secreting pituitary adenoma causing hyperthyroidism. This case is apparently unique, and challenges the present concept of dividing patients with inappropriate TSH secretion into tumour and non-tumour groups on the basis of conventional pituitary fossa radiology.

Adenoma↗