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Biomedical subjects

G I Evan

Publications and source records attributed to G I Evan.

At least 91 records · Page 5Linked to original sources
PubMed1987

Flow cytometric quantitation of DNA and c-myc oncoprotein in archival biopsies of uterine cervix neoplasia.

The c-myc nuclear associated oncoprotein has been quantitated simultaneously with DNA in nuclei extracted from archival biopsies of uterine cervix neoplasia. The oncoprotein and DNA were measured fluorimetrically in a flow cytometer using a mouse monoclonal antibody (MYC 1-6E10) and propidium iodide. Normal biopsies exhibited higher oncoprotein levels than carcinomas (P less than 0.00001). Furthermore, the maximum fluorescence signal in the normal tissue occurred at a lower antibody concentration compared with tumour tissue. There was no correlation between oncoprotein levels and histological grade, stage of disease, age of the patients or prognosis in the carcinomas. Aneuploidy, defined as a distinct second peak separate from the diploid distribution, was not a significant feature. The c-myc oncoprotein nuclear content does not appear to be a prognostic indicator in carcinoma of the cervix from the results of these studies but there is clearly diagnostic potential, particularly for automated analysis of cervical screening.

Cervix Uteri↗
PubMed1987

Abnormal distribution of c-myc oncogene product in familial adenomatous polyposis.

Monoclonal antibodies raised by synthetic peptide immunisation were used to determine the distribution of the protein product of the c-myc gene by immunocytochemical staining of archival wax embedded material from patients with familial adenomatous polyposis. Polyps from 18 cases of familial adenomatous polyposis, 10 of whom had developed malignant change, and 30 normal control colonic biopsy specimens were examined. A consistent staining pattern was observed in normal mucosa; nuclear staining in the basal proliferative zone; mixed nuclear and cytoplasmic staining in the maturation zone; and cytoplasmic localisation in the surface mature zone. In contrast, the polyps and carcinomata showed a mixed pattern of cytoplasmic and nuclear localisation in the basal proliferative zone with nuclear persistence throughout the crypts to the surface mature zone. This abnormal distribution of the c-myc oncogene product may have a role in the evolution of polyps and their subsequent malignant transformation into familial adenomatous polyposis.

Adenomatous Polyposis Coli↗
PubMed1987

Characterization of a heat shock-induced insoluble complex in the nuclei of cells.

The formation of an insoluble complex in isolated nuclei incubated at physiological temperature (37 degrees C) is demonstrated. A similar complex is shown to form in the nuclei of intact cells subjected to temperatures that induce the classical heat-shock response. The formation of this complex occurs rapidly in response to hyperthermia and is induced by small increases in temperature both in vitro and in vivo. We have characterized the formation of the complex in isolated nuclei and the nuclei of intact cells. A small number of the subset of nuclear proteins involved in the complex have been identified. The significance of the loss of solubility of these proteins in the nucleus following hyperthermia is discussed.

Cell Line↗
PubMed1987

A sensitive and quantitative enzyme-linked immunosorbence assay for the c-myc and N-myc oncoproteins.

The c-myc and N-myc nuclear oncoproteins are implicated in the genesis and maintenance of the transformed phenotype in several types of neoplastic disease, and the c-myc protein is involved in the progression of normal cells through the cell cycle. We have designed and developed sensitive and quantitative ELISAs for these proteins. Myc proteins are captured from cell lysates by an antibody directed against a peptide sequence substantially conserved in all known myc proteins; the captured proteins are recognised by a specific anti-c-myc or anti-N-myc monoclonal antibody conjugated to alkaline phosphatase; bound alkaline phosphatase is measured with an extremely sensitive cycling enzyme system that generates a coloured end-product. The c-myc assay is calibrated using bacterially expressed human c-myc protein. We have used this assay to estimate the number of c-myc molecules in a range of normal and transformed cells of human, murine, and feline origin; to monitor increases in c-myc expression when quiescent cells are stimulated with growth factors; and to follow the decrease in c-myc protein levels when HL60 promyelocytic leukaemia cells are induced to differentiate with dimethylsulphoxide or phorbol esters.

Animals↗
PubMed1986

Xenopus myc proto-oncogene during development: expression as a stable maternal mRNA uncoupled from cell division.

A Xenopus cDNA clone highly homologous to the proto-oncogene c-myc has been isolated and used to derive a homologous probe to study myc expression during embryonic development. Myc RNA is identified as a member of the class of maternal mRNAs expressed before fertilisation. It is highly accumulated from early oogenesis and an unfertilised egg contains 8 pg, about 10(5)-fold the myc content of proliferative somatic cells. After fertilisation a post-transcriptional regulation of the gene is induced and the accumulated myc RNA is degraded (t1/2 = 4 h 20 min) to reach a level at gastrula of 10 transcripts per cell; a value maintained during subsequent embryonic development. The Xenopus myc protein has also been identified by both myc-specific antibodies and hybrid selection experiments. Translation in vitro of Xenopus myc RNA shows that it encodes a 62-kd protein which is also recognised by myc antibodies in oocyte extracts. This protein is accumulated in late oogenesis. The results indicate an unusual uncoupling of myc expression and cell proliferation linked to a stabilisation of the RNA product.

Animals↗
PubMed1986

Characterization of the human c-myc protein using antibodies prepared against synthetic peptides.

Synthetic peptides with amino acid sequences based on inferred sequences encoded by the human c-myc proto-oncogene have been used as immunogens to produce polyclonal and monoclonal antibodies. These peptide-specific antibodies have then been used to identify the intact human c-myc protein. By immunoprecipitation and immunoblotting analysis we have defined the human c-myc protein as a phosphoprotein with an apparent molecular mass of 62 kDa on a sodium dodecyl sulphate-polyacrylamide gel. The protein is present in both normal and transformed cells but the steady-state levels of p62c-myc are elevated in the transformed cells by comparison with normal ones. We have used our anti-c-myc peptide antibodies to study the subcellular localization of p62c-myc. We find the protein to be loosely associated with cell nuclei by an interaction which is highly sensitive to variations in ionic strength within the physiological range. In response to in vitro or in vivo heat-shock the protein becomes sequestered in an insoluble complex associated with the nucleus. This complex contains a discrete subset of nuclear proteins, of which p62c-myc is a member. Immunofluorescence microscopy of p62c-myc shows it to be localized in a defined and previously unobserved subnuclear structure. A role for p62c-myc as an intracellular messenger is suggested.

Amino Acid Sequence↗
PubMed1986

Restricted expression of EBV latent genes and T-lymphocyte-detected membrane antigen in Burkitt's lymphoma cells.

Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing.

Antigens, Surface↗
PubMed1986

Localisation of lung cancer by a radiolabelled monoclonal antibody against the c-myc oncogene product.

A set of mouse nonoclonal antibodies against the c-myc oncogene product, a 62,000 dalton nuclear binding protein involved in cell cycle control, has been constructed by immunisation with synthetic peptide fragments. One such antibody, CT14, was radiolabelled with 131I and administered to 20 patients with different malignant diseases. Good tumour localisation was observed in 12 out of 14 patients with primary bronchial carcinoma but not in patients with pulmonary metastases from primary tumours elsewhere. Successfully localised tumours were all 3 cm or more in diameter. Monoclonal antibodies against oncogene products may provide novel selective tools for the diagnosis and therapy of cancer.

Adult↗
PubMed1986

The clinical significance of flow cytometric c-myc oncoprotein quantitation in testicular cancer.

A sensitive flow cytometric assay has been developed using a monoclonal antibody, Myc 1-6E10, to quantitate c-myc oncoprotein levels in nuclei isolated from wax embedded testicular tumours. The oncoprotein (p62c-myc) level increased significantly with increasing teratoma differentiation. Patients with intermediate and undifferentiated tumours who developed recurrence had lower p62c-myc levels than those who were disease free since their initial treatment. Such quantitative biochemical methods may provide new prognostic indices for cancer patients.

Antibodies, Monoclonal↗
PubMed1986

Orientation of the v-erb-B gene product in the plasma membrane.

The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane.

Alpharetrovirus↗
PubMed1985

A simultaneous flow cytometric assay for c-myc oncoprotein and DNA in nuclei from paraffin embedded material.

A simultaneous flow cytometric assay for the c-myc oncoprotein and DNA in nuclei extracted from archival paraffin wax embedded clinical biopsies is presented. The nuclei were extracted by pepsin digestion after dewaxing 20 micron sections. The c-myc oncoprotein was probed with a mouse monoclonal antibody. This was raised against a synthetic peptide corresponding to a hydrophilic region of the protein predicted from the amino acid sequence. The technique is illustrated with biopsies from patients with testicular cancer and with benign and malignant neoplasms of the colon.

Antibodies, Monoclonal↗
PubMed1985

Studies on the interaction of the human c-myc protein with cell nuclei: p62c-myc as a member of a discrete subset of nuclear proteins.

We have analyzed the localization of the human c-myc product (p62c-myc) at steady state in cells by immunoprecipitation and immunoblotting. We show that p62c-myc is extracted from nuclei by mild salt concentrations (below 200 mM), without affecting gross nuclear structure or causing extraction of major chromatin components. We observe no association between p62c-myc and the nuclear matrix. We also demonstrate that p62c-myc is a member of a discrete subset of nuclear proteins that are all rendered irreversibly insoluble in situ by exposure of isolated nuclei to physiological temperatures (37 degrees C). p62c-myc is sequestered into a similar insoluble complex in cells that have been subjected to heat shock. Finally, we show that avian v-myc and v-myb proteins in isolated nuclei also become insoluble after exposure to temperatures above 37 degrees C. We discuss the possible implications of these results.

Cell Fractionation↗
PubMed1985

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.

Antibodies, Monoclonal↗
PubMed1984

A simple and rapid solid phase enzyme-linked immunoadsorbence assay for screening monoclonal antibodies to poorly soluble proteins.

An ELISA is described in which protein antigens are fixed to microtitre wells. This assay is of particular use when antigens are soluble only in solutions containing SDS or other detergents, or else have been purified by preparative SDS polyacrylamide gel electrophoresis. Because the protein antigens are fixed to the microtitre wells, it is also possible to re-use antigen derivatised plates several times.

Antibodies, Monoclonal↗
PubMed1984

Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by chicken c-myb gene.

Analysis of the subcellular location of the proteins encoded by the oncogenes of avian myeloblastosis virus and avian leukemia virus E26 ( p45v -myb and p135gag -myb-ets, respectively) and by the chicken c-myb gene ( p75c -myb) shows that all three proteins are located in the nucleus. In AMV-infected (but not transformed) chicken fibroblasts p45v -myb also resides in the nucleus, indicating that a nuclear location of p45v -myb in these cells is not sufficient to achieve transformation. In AMV-transformed myeloblasts a small fraction of p45v -myb occupies an additional site in the perinuclear region of the cytoplasm. If the myeloblasts are caused to differentiate to macrophages, most of p45v -myb is found in the cytoplasm. This redistribution of p45v -myb within the cell may be responsible for reversion of the transformed phenotype.

Animals↗