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Biomedical subjects

M C Willingham

Publications and source records attributed to M C Willingham.

At least 73 records · Page 4Linked to original sources
PubMed1996

The down-regulated in adenoma (DRA) gene encodes an intestine-specific membrane glycoprotein.

The protein product of the DRA gene, a gene whose expression is down-regulated in colon adenomas and adenocarcinomas, is a membrane glycoprotein and a member of a family of sulfate transporters. It is expressed in the intestinal tract (duodenum, ileum, cecum, distal colon), but not in the esophagus or stomach. DRA mRNA expression is restricted to the mucosal epithelium, and DRA protein expression is further limited to the columnar epithelial cells, particularly to the brush border. Consistent with its expression in the differentiated columnar epithelium of the adult human colon, DRA is first expressed in the midgut of developing mouse embryos at day 16.5, corresponding with the time of differentiation of the epithelium of the small intestine. A model for the structure of the DRA protein is proposed and its possible role in colon tumorigenesis is discussed.

Animals↗
PubMed1996

Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active.

Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactose-binding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA). Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases. Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis. Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes. In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens-mediated gene transfer. Yields of ricin in soluble cell extracts were 1 microg/g in cells or, approximately, 0.1% of the total soluble protein. The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography. The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions. The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding. Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10(-12) M for tobacco ricin and 1 x 10(-11) M for castor bean ricin). The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10(-13) and 2 x 10(-12) M, respectively). The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.

Agrobacterium tumefaciens↗
PubMed1996

Ricin toxin contains three lectin sites which contribute to its in vivo toxicity.

Ricin intoxication of mammalian cells is initiated by B chain (RTB) binding to cell surface galactosides. Recombinant insect-derived RTB mutants with modifications in lectin-site subdomains 2 gamma, 1 alpha/2 gamma, and 1 alpha/1 beta/2 gamma were reassociated with plant RTA and tested for lethality in C57B1/6 6-8 weeks old mice. The LD50 of intraperitoneally injected castor bean ricin was 75 ng per 18 g mouse. The LD50 of single-site 2 gamma mutant heterodimer was 100 ng: the LD50 of the double-site 1 alpha/2 gamma mutant heterodimer was 500 ng, and the LD50 of the triple-site 1 alpha/1 beta,2 gamma mutant heterodimer was > 10 micrograms. Plant RTA alone had an LD50 of 300 micrograms. Animals died between 1 and 10 days post-injection. Histopathological examination of morbid animals receiving an LD50 dose of each toxin revealed only apoptosis in the thymus and spleen. The present data provide clear evidence for participation of three lectin sites in ricin in vivo toxicity. These results suggest an origin for some of the normal tissue toxicities observed with clinical trials of doubly blocked ricin conjugates and suggest modification of at least three RTB subdomains will be necessary in genetically engineered ricin fusion proteins.

Animals↗
PubMed1996

Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin.

Immunotoxin LMB-1 is composed of monoclonal antibody B3 chemically linked to PE38, a genetically engineered form of Pseudomonas exotoxin. B3 recognizes a carbohydrate antigen (Le(Y)) present on many human solid tumors. LMB-1 has excellent antitumor activity in nude mice bearing Le(Y)-positive tumors. We conducted a phase I study of 38 patients with solid tumors who failed conventional therapy and whose tumors expressed the Le(Y) antigen. Objective antitumor activity was observed in 5 patients, 18 had stable disease, 15 progressed. A complete remission was observed in a patient with metastatic breast cancer to supraclavicular nodes. A greater than 75% tumor reduction and resolution of all clinical symptoms lasting for more than six months was observed in a colon cancer patient with extensive retroperitoneal and cervical metastasis. Three patients (two colon, one breast cancer) had minor responses. The maximum tolerated dose of LMB-1 is 75 microgram/kg given intravenously three times every other day. The major toxicity is vascular leak syndrome manifested by hypoalbuminemia, fluid retention, hypotension and, in one case, pulmonary edema. Although immunotoxins have been evaluated in clinical studies for more than two decades, this is the first report of antitumor activity in epithelial tumors.

Adult↗
PubMed1996

Cytotoxicity of KDEL-terminated ricin toxins correlates with distribution of the KDEL receptor in the Golgi.

DNAs encoding ricin toxin A chain (RTA), with or without a C-terminal endoplasmic reticulum retention signal KDEL, were subcloned into pGEX2T bacterial expression plasmid. After transformation of JM105 E. coli cells and induction with isopropylthio-beta-galactoside (IPTG), fusion proteins were bound to an immobilized glutathione matrix and recombinant ricin A chains released with thrombin. Both recombinant wild-type RTA and RTA with KDEL had immunological reactivity and catalytic activity indistinguishable from plant RTA. The bacterial RTA products reassociated with plant ricin B chain (RTB) similarly to plant RTA. Cell cytotoxicities were measured on seven cell lines for each A-chain and heterodimer. Although KDEL sequences enhanced cytotoxicity in most cases, significant variability was observed. In each case, addition of KDEL enhanced A-chain cytotoxicity more than holotoxin cytotoxicity. Three cell lines showed reduced KDEL enhancement of both RTA and ricin cytotoxicity. The concentration of KDEL receptor was examined on each cell line by immunofluorescence microscopy with an antireceptor monoclonal antibody. Differences in sensitivity to KDEL-containing toxins correlated with altered distribution of KDEL receptor between endoplasmic reticulum (ER) and Golgi compartments.

Amino Acid Sequence↗
PubMed1996

Enhancement of radiation dose to the nucleus by vesicular internalization of iodine-125-labeled A33 monoclonal antibody.

UNLABELLED: In radioimmunotherapy, the emission characteristics of the radioisotope is critical in determining the radiation dose to the tumor compared to normal organs. If antibodies internalize and transport low-energy electron emitting isotopes close to the tumor cell nucleus, an improved therapeutic advantage is achieved. METHODS: Using fluorescent microscopy, we studied the subcellular distribution of an internalizing antibody, A33, which detects a restricted determinant on colon cancer cells. We developed a physical model to assess the dose deposited on the nucleus by electrons emitted from radiolabeled A33 accumulated inside vesicles. This model is based on the energy-range relationship of electrons in water. Similarly, another model was developed to calculate the radiation dose to the nucleus from electrons emitted from extracellular space. The percentage of A33 bound to membrane and internalized was determined in vitro at various time points. Cytotoxicity experiments were performed with 125I- and 131I-labeled A33 at various concentrations and specific activities. RESULTS: A33 accumulates in cytoplasmic vesicles (40% of total bound) which transport the activity close to the nucleus. This increases the radiation dose to the cancer cell nucleus by a factor of 3 compared to the average dose calculated based on the assumption of a uniform distribution on the cell membrane. The cytoplasm of antigen-negative normal cells shields the nucleus from the electrons emitted from extracellular 125I. This shielding is 30 times less for 131I. Cytotoxicity data show 10% cell survival with 10 microCi/ml of 125I-A33, but 90% survival with up to 100 micro/Ci/ml of 125I-A33 in the presence of a blocking dose of 100-fold excess of cold A33. Similar experiments with 131I showed cytotoxicity in both cases. CONCLUSIONS: The results of the cytotoxicity experiment are in agreement with the physical model and suggest a basis for improved tumor-to-marrow radiation dose by clinical use of 125I-A33.

Antibodies, Monoclonal↗
PubMed1996

Immunophenotype, mRNA expression, and gene structure of p53 in Wilms' tumors.

The anaplastic variant of Wilms' tumor is regarded as the result of tumor progression of the more common classic Wilms' tumor. Anaplasia is rare and occurs in only 4.5% of tumors. Three anaplastic Wilms' tumors in our collection were examined in comparison with 10 classic Wilms' tumors for p53 expression by immunohistochemical techniques and Northern blot analysis, and their p53 gene structure was determined by single-stranded conformation polymorphism and sequence analysis. All classic tumors contained a wild-type p53 gene and expressed marginal levels of protein as expected for normal p53. In contrast, three out of three anaplastic tumors demonstrated evidence of p53 alterations consistent with a role of p53 in tumor progression. One of the anaplastic mutants (W4) did not express protein or p53 mRNA. Its apparently normal immunophenotype would have disguised the mutated nature of p53, which was detected only by mRNA and sequence analysis. The second anaplastic mutant (W16) contained normal levels of p53 mRNA, but overexpressed the protein in a fashion typical of mutated p53. The same immunophenotype was displayed by fixed primary tissue of the third anaplastic tumor (W17), but p53 mutation could not be confirmed for lack of frozen primary material. The present study emphasizes the importance of p53 function in the anaplastic progression of Wilms' tumor and the risk of error in assessing normal p53 function using a single methodology.

Adolescent↗
PubMed1996

Molecular biology and the pediatric surgeon: definitions and basic methodology.

Molecular biology techniques and their application are becoming increasingly important to the practicing clinician. This article reviews the basics of DNA chemistry and highlights important molecular biology techniques. It will provide a guide for the pediatric surgeon as she/he attempts to integrate this field into everyday practice.

Genetic Techniques↗
PubMed1995

Tumor-specific anti-epidermal growth factor receptor variant III monoclonal antibodies: use of the tyramine-cellobiose radioiodination method enhances cellular retention and uptake in tumor xenografts.

Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene are characteristics of many types of tumors. One class of EGFR mutations, EGFRvIII, is characterized by an in-frame deletion resulting in a truncated external domain of the receptor. EGFRvIII was first identified in a subset of gliomas and has since been found in some non-small cell lung carcinomas and breast carcinomas. mAbs specific for this variant form of EGFR but unreactive with the wild-type EGFR have been reported from our laboratory. This study further characterizes three of these antibodies. We determined, via radiolabeling techniques and immunofluorescence microscopy, that, after cell binding in vitro, the anti-EGFRvIII-specific mAbs internalize at 37 degrees C. Furthermore, subsequent to internalization, the antibodies were processed intracellularly, presumably by lysosomal degradation. We also examined the use of an alternative radiolabeling procedure that uses nonmetabolizable radio-iodinated tyramine cellobiose. Our results show that the tyramine cellobiose labeling method allows for greater tumor cell retention of radiolabel in vitro (76% for tyramine cellobiose and 27% for Iodo-Gen after 24 h). Paired-label biodistribution studies in athymic mice indicate that anti-EGFRvIII mAb L8A4 localizes specifically to EGFRvIII-expressing tumor xenografts with a maximum of 34.3 +/- 7.6% injected dose/g when labeled using tyramine cellobiose compared with a maximum of 14.9 +/- 4.3% injected dose/g using Iodo-Gen; similar results were obtained with mAb H10. These results suggest that the anti-EGFRvIII mAbs may serve as potential carriers for radioconjugate- and immunotoxin-based therapies for tumors expressing EGFRvIII.

Animals↗
PubMed1995

Upregulation of b-FGF receptor expression after carotid bypass.

Basic fibroblast growth factor (b-FGF) appears to be an important positive modulator of the neointimal hyperplasia that occurs after prosthetic vascular graft implantation through its effects on vascular myointimal/smooth muscle cell migration and proliferation. The distribution and extent of b-FGF receptor (b-FGFR1) expression was compared using immunohistochemical techniques in normal porcine carotid arteries and at various times up to 6 weeks following implantation of small caliber prosthetic vascular grafts. At the time of graft harvest, specimens were infused with OCT medium at 100 mm Hg and rapidly frozen in liquid nitrogen. Transverse sections of the perianastomotic arterial tissues were labeled with primary mouse monoclonal antibody directed toward the extracellular domain of the receptor, followed by goat-anti mouse IgG and rabbit anti-goat IgG conjugated to horseradish peroxidase. The b-FGFR1-positive cells were identified by peroxidase activity within the Golgi complex of smooth muscle cells. Normal porcine carotid arteries showed no evidence of staining for b-FGFR1. However, at 6 weeks cells in the perianastomotic area clearly showed significant b-FGFR1 localization. Anti-muscle actin labeling confirmed these to be smooth muscle cells. The observed upregulation of b-FGFR1 expression supports the concept of positive feedback by cytokines as a contributing factor to the hyperplastic response of smooth muscle cells after prosthetic vascular graft implantation. This finding further supports a potential strategy to specifically target activated smooth muscle cells through use of mitotoxin therapy.

Animals↗
PubMed1995

Clinical trials of targeted toxins.

Immunotoxins (monoclonal antibodies chemically coupled to peptide toxins) and fusion toxins (peptide ligands fused genetically to peptide toxins) have been used to treat a variety of malignancies over the last 20 years. Problems with normal tissue toxicities (vascular leak syndrome, hepatotoxicity, and neurotoxicities), poor penetration to tumor interstitum, and humoral immune responses have limited clinical efficacy. Higher response rates were observed with systemic therapy of leukemias and lymphomas and regional therapy of primary brain tumors. Ongoing studies are examining the role of targeted toxins in combination with chemoradiotherapy and in minimal residual disease settings.

ADP Ribose Transferases↗
PubMed1994

P-glycoprotein epitope mapping. I. Identification of a linear human-specific epitope in the fourth loop of the P-glycoprotein extracellular domain by MM4.17 murine monoclonal antibody to human multi-drug-resistant cells.

A new murine monoclonal antibody (MAb), MM4.17, to human multi-drug-resistant (MDR) cells was found to be reactive in an ELISA with a synthetic 16-amino acid peptide selected from the fourth loop of the P-glycoprotein extracellular domain. Immunohistochemistry indicated that this MAb reacted in human tissues in the same pattern as that previously found with other human-specific MAbs to P-glycoprotein. For a precise definition of the MM4.17 epitope, a peptide library consisting of overlapping 4- to 10-mer residues covering the entire P-glycoprotein-fragment was synthesized on polyethylene pins and tested for MAb binding. The results of this ELISA demonstrated that the MM4.17 epitope is constituted by the continuous-linear TRIDDPET amino-acid sequence (residues 750-757 of the human MDRI-P-glycoprotein). The MAb MM4.17 recognizes only the human MDRI-P-glycoprotein isoform, and excess TRIDDPET peptide blocks the binding of the MAb to MDR variants of CEM cells. These results demonstrate that the amino-acid sequence TRIDDPET from the human MDRI gene represents the first continuous-linear epitope identified in the P-glycoprotein extracellular domain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo.

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.

ADP-Ribosylation Factor 1↗
PubMed1994

Transient mitotic phase localization of bcl-2 oncoprotein in human carcinoma cells and its possible role in prevention of apoptosis.

We used a monoclonal antibody and fluorescence immunocytochemistry to localize the product of the bcl-2 gene in two cultured human carcinoma cell lines, KB and OVCAR-3. These cells show little or no localization of bcl-2 oncoprotein in interphase cells but demonstrate a dramatic appearance of bcl-2 in early prophase or late G2, which persists through-out mitosis, rapidly disappearing at telophase. The pattern of bcl-2 localization shows a diffuse nuclear distribution before chromosome condensation, followed by a specific concentration of bcl-2 at the margins of condensed chromosomes in prophase, metaphase, and anaphase. Treatment of these cells with taxol, an agent that interfers with formation of the mitotic spindle, causes mitotic arrest and apoptosis after a prolonged incubation period. During mitotic arrest due to taxol, bcl-2 remains associated with condensed chromosomes but is lost after > 2 days at approximately the same time as the appearance of apoptotic features in these cells. Western blotting indicates that the only extractable protein reactive with this monoclonal antibody under these conditions is a approximately 28 KD form of bcl-2. These results suggest a model for the role of bcl-2 in protection from apoptosis and a potentially common mechanism by which the final step of apoptosis might be mediated.

Antibodies, Monoclonal↗
PubMed1994

High levels of p26BCL-2 oncoprotein retard taxol-induced apoptosis in human pre-B leukemia cells.

In human leukemic cells clinically relevant concentrations of taxol have been demonstrated to induce the biochemical and morphologic hallmarks of apoptosis (Leukemia 1993;7:563-568). Since overexpression of the bcl-2 gene has been reported to retard apoptosis due to a variety of anticancer agents, we examined and compared taxol-induced intracellular microtubular bundling and apoptosis in pre-B human leukemia 697 cells and their counterparts which have been transfected with and overexpress cDNA derived from the bcl-2 gene. Treatment with 0.1 or 1.0 mumol/l taxol for 24 h resulted in internucleosomal DNA fragmentation and morphologic features of apoptosis in 697 cells, but not in 697/BCL-2 cells. However, indirect immunofluorescent staining with anti-tubulin antibody revealed that taxol treatment produces stable microtubule bundles resistant to calcium-mediated disassembly in 697, as well as 697/BCL-2 cells. In addition, taxol-induced microtubule bundling was associated with a marked accumulation of the two cell types in the G2/M phase of the cell cycle. Following exposure to taxol, when 697 cells were washed and kept in drug-free medium, they showed rapid onset of apoptosis followed by loss of cell viability and a decline in cell numbers. In contrast, identically treated 697/BCL-2 cells kept in drug-free medium remained in a growth arrested state, but showed little evidence of apoptosis for up to 4 days. They eventually demonstrated features of apoptotic cell death and loss of viability between 5 and 7 days. This was not accompanied by a decrease in p26BCL-2 levels. Anti-phosphotyrosine or anti-MAP kinase immunoblot analyses of proteins isolated from taxol-treated 697 and 697/BCL-2 cells failed to show any difference in tyrosine phosphorylation of cellular proteins. Therefore, our findings indicate that in 697/BCL-2 cells, high levels of p26BCL-2 significantly delay taxol-induced endonucleolytic internucleosomal DNA fragmentation and apoptosis, but do not affect taxol-induced microtubule bundling or cell cycle growth arrest. The delayed onset of taxol-induced DNA fragmentation and apoptosis in 697/BCL-2 cells without down-regulation of p26BCL-2 levels suggests that an alternative mechanism of taxol-mediated apoptosis might be triggered which is unimpeded by high p26BCL-2 levels, or taxol-induced prolongation of mitotic arrest may lead to the inactivation or inhibition of that mechanism by which p26BCL-2 is able to block apoptosis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Light microscopy techniques for the demonstration of silicone gel.

Because of the difficulty in identifying silicone gel in histologic and cytologic specimens by means of conventional light microscopy, we investigated alternative light microscopy techniques, as well as specimen staining and preparation. Specimens from six periprosthetic capsules, one silicone granuloma specimen, and one synovial biopsy specimen obtained from women with silicone breast implants were cut at 4-, 10-, 20-, and 30-microns sections, stained with a variety of common stains, and correlated with electron probe microanalysis. In addition, a commercial silicone gel and silicone gel extracted from a previously implanted silicone breast prosthesis were smeared and examined unstained or stained with Papanicolaou and Diff Quik. Silicone was noted to be refractile, nonpolarizable, and nonstainable. Thicker sections prevented silicone "dropout" during processing and increased the contrast between stained tissue and unstained silicone (negative staining). The relative ease of silicone identification was greatly increased with non-Koehler, phase contrast, and darkfield microscopy. Staining the mounting media of hematoxylin-eosin-stained sections with a nonparticulate commercial ink allowed enhanced negative staining detection of silicone gel.

Breast↗