Search PubMed⌕ Search

Biomedical subjects

M Willingham

Publications and source records attributed to M Willingham.

15 recordsLinked to original sources
PubMed1991

B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice.

The genes encoding the heavy- and light-chain Fv regions of the monoclonal murine antibody B3, which recognizes a carbohydrate antigen on the surface of many human carcinomas, were cloned by PCR techniques and used to generate single-chain immunotoxins containing Pseudomonas exotoxin (PE). The light and heavy chains were connected by a flexible linker to form a single-chain antigen-binding protein, B3(Fv), which was in turn fused to truncated forms of PE lacking the cell-binding domain. The single-chain Fv and two different B3(Fv) immunotoxins, B3(Fv)-PE40 and B3(Fv)-PE38KDEL, were expressed in Escherichia coli and the single-chain immunotoxins were purified to near homogeneity. Both recombinant immunotoxins were shown to be cytotoxic specifically to carcinoma cell lines that express the B3 antigen on their surface; B3(Fv)-PE38KDEL was significantly more active. Furthermore, intravenous administration of B3(Fv)-PE38KDEL caused complete regression of human epidermoid carcinomas growing subcutaneously in immunodeficient mice.

ADP Ribose Transferases↗
PubMed1989

Expression of a multidrug resistance gene in human cancers.

Many cancers have been cured by chemotherapeutic agents. However, other cancers are intrinsically drug resistant, and some acquire resistance following chemotherapy. Cloning of the cDNA for the human MDR1 gene (also known as PGY1), which encodes the multidrug efflux protein P-glycoprotein, has made it possible to measure levels of MDR1 RNA in human cancers. We report the levels of MDR1 RNA in greater than 400 human cancers. MDR1 RNA levels were usually elevated in untreated, intrinsically drug-resistant tumors, including those derived from the colon, kidney, adrenal gland, liver, and pancreas, as well as in carcinoid tumors, chronic myelogenous leukemia in blast crisis, and cell lines of non-small cell carcinoma of the lung (NSCLC) with neuroendocrine properties. MDR1 RNA levels were occasionally elevated in other untreated cancers, including neuroblastoma, acute lymphocytic leukemia (ALL) in adults, acute nonlymphocytic leukemia (ANLL) in adults, and indolent non-Hodgkin's lymphoma. MDR1 RNA levels were also increased in some cancers at relapse after chemotherapy, including ALL, ANLL, breast cancer, neuroblastoma, pheochromocytoma, and nodular, poorly differentiated lymphoma. Many types of drug-sensitive and drug-resistant tumors, including NSCLC and melanoma, contained undetectable or low levels of MDR1 RNA. The consistent association of MDR1 expression with several intrinsically resistant cancers and the increased expression of the MDR1 gene in certain cancers with acquired drug resistance indicate that the MDR1 gene contributes to multidrug resistance in many human cancers. Thus, evaluation of MDR1 gene expression may prove to be a valuable tool in the identification of individuals whose cancers are resistant to specific agents. The information may be useful in designing or altering chemotherapeutic protocols in these patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

The E5 oncoprotein of bovine papillomavirus is oriented asymmetrically in Golgi and plasma membranes.

The E5 oncoprotein of BPV-1 is a 44 amino acid, hydrophobic polypeptide which is present in membrane fractions of transformed cell homogenates. To define the specific cellular membrane compartments with which E5 associates, immunofluorescence and immunoelectron microscopy were performed using an affinity-purified antibody specific for the E5 carboxyl-terminus. Two cell systems which overexpressed the E5 protein were analyzed: (1) Balb/c 3T3 mouse cells transformed by a BPV-1 cDNA expressing the E2-E5 ORFs, and (2) Sf9 insect cells infected with recombinant baculovirus expressing the E5 ORF. In both cell types, E5 protein was found in the membranes of the Golgi apparatus with its carboxyl-terminus oriented intraluminally. In the Sf9 cells, which synthesize much greater quantities of viral protein, E5 protein was observed additionally in plasma membranes with the carboxyl-terminus facing extracellularly. The concentration of E5 protein within the Golgi and plasma membranes suggests several potential mechanisms for inducing cellular transformation.

Amino Acid Sequence↗
PubMed1988

Purification of transiently transfected cells by magnetic affinity cell sorting.

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

Animals↗
PubMed1986

A mouse tumor-specific transplantation antigen is a heat shock-related protein.

A tumor-specific transplantation antigen has been purified to homogeneity from the cytosol of a methylcholanthrene-induced tumor, Meth A. The purified antigen is highly immunogenic and specific against challenge with Meth A, providing greater than 95% inhibition of tumor growth in immunized syngeneic mice. Immunofluorescence analysis of Meth A showed that the antigen is a highly abundant cytosolic protein but that it is also present at the cell surface and, therefore, accessible to the host's immune system. The antigen consists of two polypeptide isoforms present in equimolar amounts, having similar masses (84 and 86 kDa), pI values (4.95 and 4.90), and amino acid compositions. Both are phosphoproteins, and neither is glycosylated. The NH2-terminal sequences of the two isoforms are identical except that each chain contains a portion of unique sequence. Comparison of the NH2-terminal and CNBr-fragment sequence data to the sequences of the yeast and Drosophila heat shock proteins (Hsp90 and Hsp83, respectively) reveals that 73 of 91 residues compared are identical. In addition, an anti-Meth A tumor antigen serum that defects the isoforms from a variety of tumors also immunoprecipitates proteins of identical mass and pI from both normal and heat-shocked mouse embryo cells.

Amino Acid Sequence↗
PubMed1978

Cellular transformation and the 'morphologic phenotype' of transformed cells.

Expression of the product of the transforming gene (src) of RNA tumour viruses promotes growth and usually alters the adhesion, appearance and surface properties of cultured fibroblasts. The latter group of properties termed the 'morphologic phenotype' of transformed cells is largely due to diminished cell-to-substratum adhesion. The role of cyclic AMP, cell surface protein (CSP), and other factors in producing the 'morphologic phenotype' are discussed. The effects of src expression bear a striking resemblance to the action of peptide hormones such as insulin on appropriate target cells.

Animals↗
PubMed1978

Cyclic AMP-dependent phosphorylation of the actin-binding protein filamin.

Filamin is a high molecular weight protein that binds to actin filaments in cells. It is found in large amounts in several different cells and tissues, including smooth muscle, fibroblasts, platelets, and macrophages. It is immunologically related to the previously described macrophage high molecular weight actin-binding protein but clearly different from erythrocyte spectrin. Filamin is a phosphoprotein; it is phosphorylated in vivo in intact tissues and cells. It can be phosphorylated in vitro with endogenous kinases; cyclic AMP stimulates this phosohorylation. Furthermore, the purified protein can be phosphorylated by purified cyclic AMP-dependent protein kinase. In smooth muscle homogenates, the stimulation of filamin phosphorylation by cyclic AMP is specific. Cyclic GMP and Ca2+ do not increase its phosphorylation, although they do stimulate phosphorylation of other proteins.

Actins↗
PubMed1977

Localization of serum-derived alpha 2 macroglobulin in cultured cells and decrease after Moloney sarcoma virus transformation.

NRK cells and many other cultured fibroblasts were found to contain the protease inhibitor, alpha 2 macroglobulin (alpha 2M). This alpha 2M is present as a result of uptake of alpha 2M from the calf serum in the culture medium. Some of this alpha 2M is released back into the medium. In radiolabeling experiments with 14C-amino acids, no radioactivity was detected in intracellular or extracellular alpha 2M. Fluorescence microscopy of fixed cells using rhodamine-labeled antibodies indicated that alpha 2M is present in vesicular organelles different from primary lysosomes. Fluorescence microscopy of living cells shows that rhodamine-labeled alpha 2M (rhodamine-alpha 2M) is taken up into similar structures. Of the many cell lines examined, Moloney sarcoma virus-transformed cells had the lowest amounts of alpha 2M. Some of the effects of serum on the behavior of cultured cells could be a consequence of inhibition of cellular proteases by alpha 2M.

Animals↗
PubMed1977

Role of cell surface carbohydrates and proteins in cell behavior: studies on the biochemical reversion of an N-acetylglucosamine-deficient fibroblast mutant.

AD6, a mutant derived from 3T3 Balb/c cells, is characterized by low adhesion to substratum, round shape, increase in surface microvilli, increase in agglutinability by concanavalin A, and loss of directional motility. These properties are often observed in transformed cells. However, the mutant has normal growth properties and anchorage-dependence of growth, and it does not form tumors. In AD6, the biosynthesis of complex carbohydrates and glycoproteins is impaired because of a block in the acetylation of GlcN-6-P. This defect is responsible for all the surface alterations because feeding of GlcNAc to AD6 cells corrects the defects in the synthesis of complex carbohydrates and the exposure of glycoproteins at the outer surface of the plasma membrane. Parallel to this biochemical reversion, there is full restoration of the altered biological properties. In contrast, GlcNAc has no effect on the morphologic features of two lines of transformed cells. Our results suggest that the carbohydrate portion of cell surface proteins has an important role in adhesion and related aspects of cell behavior. The fact that a defined alteration of the cell surface induces many properties often encountered in transformed cells, without affecting control of cell division, strongly suggests that these alterations in properties are not sufficient to account for the loss of growth regulation.

Acetylglucosamine↗
PubMed1976

Cell surface myosin in cultured fibroblasts.

The surface components of L-929 mouse fibroblast cells in monolayer culture have been labeled with 125I by lactoperoxidase-catalyzed iodination procedure. One of the membrane proteins iodinated has been shown to be myosin as follows: it has the same electrophoretic mobility (molecular weight 200,000 daltons) as myosin heavy chain on sodium dodecylsulfate polyacrylamide gels and the labeled myosin is specifically precipitated by fibroblast myosin antiserum from a preparation of purified plasma membrane that have been solubilized by treatment with 1% Triton X-100. One other 125I-labeled membranes protein (molecular weight 210, 000 daltons) is precipitated along with myosin; the latter does not combine directly with antimyosin antibody. This was determined by reacting polyacrylamide gels containing the separated membrane proteins with fibroblast myosin antiserum; myosin was the only membrane protein reacting with the antibody as determined by two separate methods. Membrane myosin is not labeled when the cells are grown in 14C-D-glucosamine or treated with galactose oxidase and potassium borotritide. Thus membrane myosin is probably not a glycoprotein.

Cell Membrane↗
PubMedDate not supplied

Antitumor activity of a thioether-linked immunotoxin: OVB3-PE.

A thioether-linked immunotoxin was made between Pseudomonas exotoxin and the monoclonal antibody OVB3. This conjugate, OVB3-PE, was cytotoxic for the human ovarium cancer cell line OVCAR-3 (ID of 2.5 x 10(-12) M) and it was therefore tested for antitumor activity in a nude mouse model of ovarian cancer. This model employs the injection of a lethal number of OVCAR-3 cells into the peritoneal cavity of nude mice. When 0.2-1 micrograms of OVB3-PE was injected intraperitoneally on three successive days beginning 3-5 days after OVCAR-3 cell implantation, the survival of the tumor-bearing mice was increased 2-4-fold compared to that of untreated control mice. Median survival times for control mice ranged from 44 to 50 days while survival times of 150 days or greater were seen in mice treated with OVB3-PE. When OVB3-PE administration was delayed until 2-4 weeks after tumor cell implantation, OVB3-PE treatment also showed antitumor activity, but the duration of survival was less than with the early treatments. OVB3-PE was also cytotoxic for MCF-7 breast carcinoma cells, HT-29 colon carcinoma cells, and A431 epidermoid carcinoma cells.

Animals↗