Biomedical subjects

M C Willingham

Publications and source records attributed to M C Willingham.

At least 109 records · Page 6Linked to original sources

PubMed1991

Immunohistochemical analysis of p53 and HER-2/neu proteins in human tumors.

We examined samples of tumors of human breast, ovary, and colon of various degrees of malignancy for the expression of p53 protein, using a panel of anti-p53 antibodies and peroxidase immunohistochemistry. Of 66 tumor cases (24 cases of ovarian carcinoma, 23 cases of colon adenocarcinoma, and 19 cases of breast carcinoma), 36 (53%) showed high levels of expression of p53 using a human-specific antibody, and 16 (24%) showed high expression of a mutant form of p53. In the mutant p53-positive breast tumor samples, six (86%) were positive for HER-2/neu reactivity, compared with colon (0/4) and ovarian tumors (1/5). The pattern of p53 intracellular localization and tissue distribution, and the relationship between the expression of mutant p53 and cell differentiation, were also examined; poorly differentiated cells showed either overexpression of p53 or higher levels of mutant p53 in comparison with more normal cells.

Breast Neoplasms↗

PubMed1991

Intracellular distribution of the endogenous and transfected beta form of thyroid hormone nuclear receptor visualized by the use of domain-specific monoclonal antibodies.

To study the regulation, tissue distribution, and subcellular localization of nuclear receptor for thyroid hormone, monoclonal antibodies (mAbs) against the human placental c-erbA (hTR beta 1) protein were prepared. hTR beta 1 was expressed in Escherichia coli and purified to apparent homogeneity. The purified hTR beta 1 was used to produce monoclonal antibodies. Three hybridomas, secreting mAb J51, J52, and J53, were isolated. All of these mAbs recognized hTR beta 1. J51 and J52 belong to the immunoglobulin G1-k subclass; J53 is an IgM. To evaluate cross-reactivity with other classes of c-erbAs, the three mAbs were used to immunoprecipitate the in vitro translation products of human (h) TR alpha 1, TR alpha 2, rat (r) TR beta 1, TR alpha 1, and TR alpha 2. None of these three mAbs reacted with h- or rTR alpha 1 and TR alpha 2. J51 did not react with rTR beta 1, but J52 and J53 cross-reacted with rTR beta 1 with the same activity as hTR beta 1. To localize the epitopes in the hTR beta 1 molecule, [35S]methionine-labeled and truncated hTR beta 1 containing the hormone-binding domain E (Lys235-Asp456; Lys201-Pro414), domain D (Met169-Asp456), or the DNA-binding domain C (Glu100-Asp456) were expressed in E. coli and purified. Immunoprecipitation of the above truncated hTR beta 1 with mAbs indicated that the epitopes for J51 and J52 were located in two different sites in the A/B domain. The epitope for J53 was located in the E domain. Using immunocytochemistry and mAb J52, the endogenous TR beta 1 in rat pituitary GH3 cells was visualized to be exclusively present in nuclei. The transfected hTR beta 1 in monkey COS-1 and human choriocarcinoma JEG-3 cells was recognized by both J51 and J52. Interestingly, the intracellular localization of the transfected hTR beta 1 or rTR beta 1 in the above two cell lines depended on the level of expression. TR beta 1 expressed at low levels was found exclusively in nuclei. However, for high level expression of TR beta 1, cytoplasmic localization was also detected. J53, however, failed to detect nuclear fluorescence of the endogenous and transfected TR beta 1 in fixed cells, suggesting that its antigenic site might be occluded. Localization of the endogenous and transfected TR beta 1 in nuclei indicated that these two receptor proteins are structurally indistinguishable.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Fluorescent verapamil derivative for monitoring activity of the multidrug transporter.

Multidrug resistance to amphipathic natural product chemotherapeutic drugs is conferred on cancer cells by expression of the MDR1 gene, which encodes the 170-kDa multidrug transporter known as P glycoprotein. The P glycoprotein-mediated efflux of toxic chemotherapeutic drugs can be reversed by agents such as verapamil, which is a substrate for the multidrug transporter and appears to be a competitive inhibitor of the efflux pump. In this study, Bodipy-verapamil, a fluorescent derivative of verapamil, has been shown to be a substrate for the efflux pump activity of P glycoprotein. Single-cell fluorescence analysis reveals that Bodipy-verapamil accumulates in lysosomes of drug-sensitive NIH3T3 and KB cells but is rapidly effluxed from multidrug-resistant derivatives of these cell lines. Although Bodipy-verapamil is a substrate for the multidrug transporter, it is not an efficient inhibitor of the pump and does not reverse resistance to vinblastine and colchicine as effectively as does verapamil. This new derivative may be a useful tool for imaging of lysosomes in drug-sensitive cells and for rapid screening for the multidrug-resistant phenotype in other cell types.

3T3 Cells↗

PubMed1990

IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells.

A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases. Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R. Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R. The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R. Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice. Similarly, the growth of an aggressive s.c. solid tumor could also be inhibited. Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin. Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor. Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.

ADP Ribose Transferases↗

PubMed1990

Expression of the human multidrug transporter in insect cells by a recombinant baculovirus.

The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. We expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [3H]-azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents (e.g., daunomycin greater than verapamil greater than vinblastine approximately vincristine) compete with the [3H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

The recombinant immunotoxin anti-Tac(Fv)-Pseudomonas exotoxin 40 is cytotoxic toward peripheral blood malignant cells from patients with adult T-cell leukemia.

Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin containing the heavy and light variable regions of the anti-Tac monoclonal antibody fused to a mutant form of Pseudomonas exotoxin (PE). Anti-Tac binds to the p55 subunit of the human interleukin 2 (IL-2) receptor, and anti-Tac(Fv)-PE40 kills human or monkey cell lines that contain either the intact IL-2 receptor or its p55 subunit alone. To assess the usefulness of anti-Tac(Fv)-PE40 in treatment of IL-2 receptor-positive leukemia, we tested peripheral blood mononuclear cells from six patients with adult T-cell leukemia. In each of the six patients, anti-Tac(Fv)-PE40 was extremely cytotoxic to the malignant cells. Metabolic activity and sensitivity of the fresh cells improved when a small amount of IL-2 (10 units per ml) was present during incubation. The toxin concentration necessary to inhibit protein synthesis by 50% after 16-hr incubation of cells with immunotoxin varied from 1.6 to 16 ng/ml (2.5-25 x 10(-11) M). In every case, binding was by means of the Tac antigen because anti-Tac(Fv)-PE40 cytotoxicity was prevented by adding excess anti-Tac antibody. Moreover, anti-Tac alone or an inactive mutant of anti-Tac(Fv)-PE40 without ADP-ribosylation activity had very little cytotoxic activity. Peripheral blood mononuclear cells from normal controls, from a patient with Tac-negative leukemia, and from adult T-cell leukemia patients without significant peripheral blood involvement were not sensitive to anti-Tac(Fv)-PE40. These results indicate that anti-Tac(Fv)-PE40 is a potent cytotoxin against adult T-cell leukemia cells in vitro and warrants clinical testing.

ADP Ribose Transferases↗

PubMed1990

A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.

We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxins were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.

Amino Acid Sequence↗

PubMed1990

ADP-ribosylation factor is functionally and physically associated with the Golgi complex.

ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.

ADP-Ribosylation Factor 1↗

PubMed1990

Activity of the multidrug transporter results in alkalinization of the cytosol: measurement of cytosolic pH by microinjection of a pH-sensitive dye.

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.

Alkalies↗

PubMed1990

A reversible multi-well chamber for incubation of cultured cells with small volumes: application to screening of hybridoma fusions using immunofluorescence microscopy.

Immunofluorescence microscopy is a powerful technique for detecting the location of surface and intracellular antigens in individual cells. However, using standard methods, processing large numbers of samples for immunofluorescence is cumbersome and difficult. To simplify greatly this process, we have developed a chamber that reversibly creates multiple small wells in a large (150 mm) tissue culture dish. This device allows the rapid and convenient processing of hundreds of samples each of 100 microliters volume. Each sample is examined using a short working distance, high numerical aperture immersion objective for maximum sensitivity and resolution. This apparatus makes immunofluorescence a practical method for the primary screening of hybridoma clones.

Antibodies, Monoclonal↗

PubMed1989

Domain II mutants of Pseudomonas exotoxin deficient in translocation.

Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.

ADP Ribose Transferases↗

PubMed1989

Enhancement of the activity of immunotoxins by analogues of verapamil.

Verapamil has been shown to enhance immunotoxin activity but only at concentrations that are too high for in vivo use. Therefore, four structural analogues of verapamil (D792, D595, D528, and Sz45) were evaluated for their ability to enhance the in vitro activity of immunotoxins made with either ricin A chain or Pseudomonas exotoxin. The following immunotoxins were used: HB21-PE and 454A12-rRTA which recognize the human transferrin receptor; and 260F9-rRTA which reacts with human ovarian carcinoma and breast carcinoma cells. The activities of the immunotoxins were determined in ovarian carcinoma cells and in KB cells using inhibition of either protein synthesis or colony formation as a measure for the cytotoxicity of the immunotoxins. Each of the four analogues enhanced the activity of ricin A-immunotoxins in a dose-dependent manner. D792 and D595 also increased the activity of IIB21-PE. Low concentrations of either Sz45 or D528 enhanced the activity of HB21-PE, but high concentrations of these two analogues either had less enhancing potency than low concentrations or even decreased the activity of HB21-PE. Specificity of enhancement by the analogues was shown by competition of the activity of the immunotoxins by the corresponding antibody and by inactivity of an irrelevant immunotoxin. The amount of enhancement ranged from 2-fold to greater than 60-fold and was dependent on the cell line and on the experimental conditions. The enhancing ability of the drugs did not correlate with their calcium-antagonistic activity. When compared with verapamil, D792 and D595 had greater enhancing potency with regard to both ricin A-immunotoxins and Pseudomonas exotoxin-immunotoxins. Greater enhancing potency and less in vivo toxicity makes D792 a candidate for use in the enhancement of immunotoxins in vivo.

ADP Ribose Transferases↗

PubMed1989

Deletion and insertion mutants of the multidrug transporter.

The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1989

Chemoimmunotoxin therapy against a human colon tumor (HT-29) xenografted into nude mice.

The efficacy of intracavitary chemoimmunotoxin therapy for cancer treatment was evaluated using the human colon carcinoma (HT-29) which had been xenografted i.p. into nude mice. Mice bearing HT-29 were treated with an immunotoxin consisting of the monoclonal antibody OVB3 coupled to Pseudomonas exotoxin (OVB3-PE), with cyclophosphamide (Cy), or with both OVB3-PE plus Cy. Mice given injections i.p. of 3 x 10(6) HT-29 ascites cells developed a localized disease that presented as both malignant ascites and solid tumor confined to the peritoneal cavity. All mice died within 30 to 40 days. Mice that received either three or six injections of OVB3-PE at a dose of 0.5 micrograms every other day beginning 3 days post-tumor inoculation exhibited significantly increased median survival times (MSTs) (P = 0.002) of 62 and 68 days, respectively, as compared to a MST of 33 days for the controls. OVB3 alone or an irrelevant monoclonal antibody conjugated to PE exhibited no antitumor activity. The therapeutic effects of the immunotoxin could be blocked by giving a large amount of unconjugated OVB3 at the same time. Treatment of mice with Cy alone at the maximal tolerated dose (250 mg/kg) on Days 10 and 17 after tumor inoculation increased the MST from 33 days to 54 days. The maximum tolerated dose could be increased to 300 mg/kg per injection if the Cy treatment was preceded by 100 mg/kg of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721), a sulfhydryl compound that selectively protects normal tissue against the toxicity of radiation and alkylating agents. Cy plus WR-2721 treatment on Days 10 and 17 increased the MST from 35 to 61 days (P = 0.002). Interestingly, groups of mice that received either two, four, or seven treatments of OVB3-PE following Cy plus WR-2721 therapy exhibited a further increase (P less than 0.002) in MSTs to 81, 87, and 96 days, respectively. Thus, the combination of cytoreductive chemotherapy with the OVB3-PE was significantly more effective for the intracavitary treatment of established HT-29 colon cancer xenografts than either chemotherapy or immunotoxin therapy alone.

ADP Ribose Transferases↗

PubMed1989

A cytoplasmic thyroid hormone binding protein: characterization using monoclonal antibodies.

We have previously purified a cellular thyroid hormone binding protein (p58) from a human carcinoma cell line [Kitagawa, S., Obata, T., Hasumura, S., Pastan, I., & Cheng, S.-y. (1987) J. Biol. Chem. 262, 3903-3908]. In the present study, the binding characteristics, the molecular properties, and subcellular localization of p58 were further characterized. Binding of the purified p58 to thyroid hormones was examined. Analysis of binding data indicates that p58 binds to 3,3',5-triiodo-L-thyronine (T3) with a Kd of 24.3 +/- 0.3 nM and n = 0.71. p58 binds to L-thyroxine similarly as to T3. However, D-T3 and reverse-T3 bind to p58 with an affinity 4- and 20-fold less than that of T3, respectively. By use of the purified p58 as an immunogen, two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated; both antibodies belong to the IgG1K subclass. J12 recognizes p58 from human, monkey, dog, hamster, and rat, but not mouse. J11 exhibits a similar species specificity except that it does not react with p58 from hamster. With these antibodies, p58 was found to be not posttranslationally modified by glycosylation, sulfation, or phosphorylation. It has a cellular degradation rate t1/2 congruent to 2.1 h. Immunocytochemical studies indicate that p58 is located in the nonmembranous cytoplasm (cytosol). These results are consistent with subcellular fractionation studies which show that greater than 95% of J11 and J12 reactivity and T3 binding activity can be found in the 110,000g supernatant.

Animals↗

PubMed1989

Antitumor activity in mice of an immunotoxin made with anti-transferrin receptor and a recombinant form of Pseudomonas exotoxin.

LysPE40 is a modified form of Pseudomonas exotoxin that lacks the cell-binding domain and has a chemically reactive lysine residue near the amino terminus. LysPE40 is made in Escherichia coli and secreted into the medium from which it is readily purified. Two immunotoxins were constructed by coupling LysPE40 to an antibody to the human transferrin receptor (TFR) or to an antibody to the human interleukin-2 receptor. These immunotoxins were selectively cytotoxic to receptor-bearing cells in tissue culture. Anti-TFR-LysPE40 given intraperitoneally to mice appeared rapidly in the blood and caused regression of A431 tumors growing as subcutaneous xenografts. These results show that it is possible to cause regression of a solid carcinoma by an immunotoxin if proper targeting can be achieved.

ADP Ribose Transferases↗

PubMed1989

Cardiac allograft survival in mice treated with IL-2-PE40.

IL-2-PE40 is a chimeric protein composed of human interleukin 2 (IL-2) genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain. IL-2-PE40, which is extremely cytotoxic to IL-2 receptor-positive cells, was examined for its ability to prevent graft rejection in mice in which activation of T cells is prominent. We demonstrate that intraperitoneally administered IL-2-PE40 specifically and significantly prolongs the survival of vascularized heart allografts in mice. The chimeric toxin, IL-2-PE40, offers an alternative approach to the treatment of autoimmune diseases and transplant rejection in humans.

ADP Ribose Transferases↗

PubMed1989

Expression of a human multidrug resistance cDNA (MDR1) in the bone marrow of transgenic mice: resistance to daunomycin-induced leukopenia.

The human multidrug resistance gene (MDR1) encodes a drug efflux pump glycoprotein (P-glycoprotein) responsible for resistance to multiple cytotoxic drugs. A plasmid carrying a human MDR1 cDNA under the control of a chicken beta-actin promoter was used to generate transgenic mice in which the transgene was mainly expressed in bone marrow and spleen. Immunofluorescence localization studies showed that P-glycoprotein was present on bone marrow cells. Furthermore, leukocyte counts of the transgenic mice treated with daunomycin did not fall, indicating that their bone marrow was resistant to the cytotoxic effect of the drug. Since bone marrow suppression is a major limitation to chemotherapy, these transgenic mice should serve as a model to determine whether higher doses of drugs can cure previously unresponsive cancers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗