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M C Willingham

Publications and source records attributed to M C Willingham.

At least 37 records · Page 2Linked to original sources
PubMed1999

Toxicology and pharmacokinetics of DTGM, a fusion toxin consisting of a truncated diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor, in cynomolgus monkeys.

We developed a fusion toxin consisting of the catalytic and translocation domains of diphtheria toxin linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF) (DTGM) for the treatment of patients with acute myeloid leukemia (AML). Our goal in this study was to determine the toxicity and pharmacokinetics of DTGM in cynomolgus monkeys (Macacca fascicularis), which possess cross-reactive GM-CSF receptors. Four groups of young adult monkeys (6 males and 12 females) were treated with five daily bolus iv infusions of 1, 5, 7.5, and 10 microgram/kg DTGM. Monkeys (2 males and 2 females) treated at 1 microgram/kg/day showed no significant side effects. Monkeys (2 males and 2 females) treated at 5 microgram/kg/day showed Grade 1-2 thrombopenia (NCI common toxicity criteria) on day 9. In contrast, monkeys (6 females) treated at 7.5 microgram/kg/day developed Grade 3 neutropenia, Grade 1-2 thrombopenia, Grade 1-3 anemia, and Grade 1-3 hypoalbuminemia. The neutropenia developed by day 4 in the 7.5 microgram/kg/day monkeys and by day 3 or 5 in the 10 microgram/kg/day monkeys and resolved in both groups by day 9, but the thrombopenia, anemia, and hypoalbuminemia persisted until day 16. Monkeys (2 male and 2 female) treated with 10 microgram/kg/day showed Grade 4 neutropenia that resolved by day 8 and Grade 2-3 anemia, hypoalbuminemia, and thrombopenia. Three of the animals developed sepsis. DTGM plasma half-life was 30 min with a peak concentration of 0.1 microgram/mL or 2 nM (1000-fold higher than the IC50 in vitro for AML blasts). Immune responses were minimal in all animals tested at 14 and 28 days with anti-DTGM levels <1 microgram/mL. All four animals at 10 microgram/kg died or were euthanized, and necropsies were performed. Animals necropsied on days 4 and 6 showed marked apoptosis and hypoplasia in the marrow, which was completely resolved for animals necropsied on day 9. No injury to other organs, including kidney, heart, liver, central nervous system, or lung, was seen. The drug was selectively toxic to malignant or differentiated myeloid cells with little toxicity to myeloid progenitors or other organs. Minimal effects in nontarget tissues make DTGM a promising candidate chemotherapeutic agent.

Animals↗
PubMed1999

Vitaxin, a humanized monoclonal antibody to the vitronectin receptor (alphavbeta3), reduces neointimal hyperplasia and total vessel area after balloon injury in hypercholesterolemic rabbits.

The vitronectin receptor (alphavbeta3) mediates several biological processes that are critical to the formation of a neointima after coronary interventions. Blockade of alphavbeta3 could reduce neointima formation by inhibiting smooth muscle cell migration, decreasing transforming growth factor-beta1 expression, enhancing apoptosis, or reducing neovasculature. The effects of short-term administration of Vitaxin, a humanized monoclonal antibody to alphavbeta3, on the responses to balloon injury were tested in hyperlipidemic rabbits. Balloon angioplasty was performed on the iliac arteries of male New Zealand White rabbits that were fed an atherogenic diet for 1 week before injury and until euthanization at 4 weeks. Rabbits were given either saline (control) or 1 of 2 dosing regimens of Vitaxin (high dose, 5.0 mg/kg, and low dose, 0.5 mg/kg), which were administered intra-arterially before injury and intramuscularly on days 2 and 3. High-dose and low-dose Vitaxin were equally effective in decreasing neointima formation even in the presence of hypercholesterolemia, a stimulus to alphavbeta3 expression. Vitaxin reduced transforming growth factor-beta1 and enhanced apoptosis in injured arteries. Despite these positive effects, Vitaxin administration was associated with a reduction in artery size, indicating a negative effect on remodeling. Vitaxin has a potential role in preventing intimal hyperplasia, especially if the negative effects on remodeling can be overcome, by dose adjustment or other strategies.

Angioplasty, Balloon↗
PubMed1999

Modified LDLs induce and bind to membrane ruffles on macrophages.

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL), or beta-migrating very low density lipoprotein (betaVLDL), a naturally occurring lipoprotein. Incubation ofmacrophages with AcLDL and OxLDL resulted in stimulation of membrane ruffle formation, while betaVLDL primarily resulted in increased numbers of microvilli. Time-lapse Allen video enhanced contrast differential interference contrast (AVEC-DIC) light microscopy and correlative whole mount intermediate-voltage transmission electron microscopy (IVEM) was used to examine the dynamics ofAcLDL stimulated membrane ruffling and membrane ruffle ultrastructure. Stereo 3D surface replicas confirmed that AcLDL bound to these AcLDL-induced membrane ruffles. Quantification of the plasma membrane surface area after incubation with AcLDL, betaVLDL or LDL confirmed that AcLDL stimulated membrane ruffling, while betaVLDL and LDL stimulated microvilli formation. These studies suggest that modified LDLs induce circular membrane ruffles and modified LDLs bind to these ligand-induced membrane ruffles.

Animals↗
PubMed1999

Modified LDLs are internalized by macrophages in part via macropinocytosis.

Macrophage foam cell formation in vitro requires uptake of modified low density lipoproteins (LDL) such as acetylated LDL (AcLDL) and moderately oxidized LDL (OxLDL). Macrophages incubated with AcLDL and OxLDL, but not LDL, showed increased membrane ruffling as seen with time-lapse phase contrast video light microscopy. Modified LDLs stimulated circular membrane ruffles between 2 and 10 min after incubation. These membrane ruffles were readsorbed into the plasma membrane between 5 and 15 min later. Phase-bright macropinosomes formed at the base of the stimulated membrane ruffles. The fluid-phase marker lucifer yellow labeled the modified LDL stimulated macropinosomes. Modified LDLs stimulate fluid-phase uptake by 1.5-fold to threefold as measured with 14C-sucrose uptake. Transmission electron microscopy showed that gold conjugated AcLDL and OxLDL bound preferentially to membrane ruffles and were present in macropinosomes (diameter >0.2 pm) underneath these membrane ruffles. AcLDL and OxLDL were also present in clathrin-coated pits and endosomes. These studies suggest that modified lipoproteins stimulate macropinocytosis. AcLDL and OxLDL are partially internalized by macropinocytosis and partially internalized via clathrin-coated pit endocytosis.

Animals↗
PubMed1999

Prognostic significance of Bcl-2 in Wilms' tumor and oncogenic potential of Bcl-X(L) in rare tumor cases.

Anaplastic Wilms' tumors are commonly believed to be rare forms of progression, driven by p53 mutations, of the more common classical Wilms' tumor or nephroblastoma Contrary to classical Wilms' tumors, anaplastic tumors traditionally tend to metastasize, to be drug-resistant and to have a poor prognosis. The Bcl-2 gene product protects cells from programmed cell death, and its over-expression has been proposed to be tumorigenic and to mediate resistance to therapy. Because Bcl-2 has been reported to be transcriptionally repressed by p53, using immuno-histochemistry and mRNA analyses, we have examined Bcl-2 expression in a panel of 10 classical and 3 anaplastic nephroblastomas in which the p53 status had been previously analyzed. We found that classical Wilms' tumors expressed significant amounts of Bcl-2 mRNA and protein, whereas anaplastic tumors did not, regardless of p53 mutations. However, because mortality occurred both among patients with classical and among those with anaplastic tumors, which had divergent Bcl-2 expression, analysis of variance failed to demonstrate prognostic Bcl-2 significance. Therefore, we examined the expression of the Bcl-X and Bax genes, which are known to synergize and antagonize Bcl-2, respectively. With the exception of anaplastic tumor W17, the monotony of Bcl-X and Bax mRNA levels did not suggest that the expression of these apoptosis-regulating genes could have a role in the prognosis of nephroblastoma. In addition to the standard 2.7-kb Bcl-X(L) mRNA, W17 expressed a 3.5-kb mRNA species which had the same coding capacity for Bcl-X(L) as the 2.7-kb mRNA. Western analysis demonstrated that W17 had the highest level of Bcl-X(L) protein, suggesting that Bcl-X(L) over-expression could play a part in the development of anaplasia in rare Wilms' tumor cases without affecting prognosis.

Blotting, Northern↗
PubMed1999

p53/p21(WAF1/CIP1) expression and its possible role in G1 arrest and apoptosis in ellagic acid treated cancer cells.

Ellagic acid is a phenolic compound present in fruits and nuts including raspberries, strawberries and walnuts. It is known to inhibit certain carcinogen-induced cancers and may have other chemopreventive properties. The effects of ellagic acid on cell cycle events and apoptosis were studied in cervical carcinoma (CaSki) cells. We found that ellagic acid at a concentration of 10(-5) M induced G arrest within 48 h, inhibited overall cell growth and induced apoptosis in CaSki cells after 72 h of treatment. Activation of the cdk inhibitory protein p21 by ellagic acid suggests a role for ellagic acid in cell cycle regulation of cancer cells.

Apoptosis↗
PubMed1999

Apoptotic cell death induced by baccatin III, a precursor of paclitaxel, may occur without G(2)/M arrest.

PURPOSE: Paclitaxel has been demonstrated to possess significant cell-killing activity in a variety of tumor cells by induction of apoptosis, but the mechanism by which paclitaxel leads to cell death and its relationship with mitotic arrest is not entirely clear. In this study, baccatin III, a synthetic precursor of paclitaxel, was used to analyze whether paclitaxel-induced apoptosis can be a separate event from microtubule bundling and G(2)/M arrest. METHODS: Several different methods including DNA fragmentation, flow cytometric analyses, TdT-mediated dUTP nick end labeling (TUNEL) and time-lapse video microscopy were used to analyze apoptotic cell death induced by baccatin III and its possible correlation with cell cycle distribution. RESULTS: Our results demonstrated that baccatin III could also cause apoptotic cell death in both BCap37 (a human breast cancer cell line) and KB cells (derived from human epidermoid carcinoma), but had less effect on microtubule bundling and G(2)/M arrest. Furthermore, we demonstrated that most apoptotic events induced by baccatin III were not coupled with G(2)/M arrest. Instead, these apoptotic events occurred predominantly in the cells in other phases of the cell cycle. CONCLUSION: Baccatin III, which contains the core taxane ring, is the fundamental piece of paclitaxel structure. The finding of baccatin III-induced apoptosis independent of cell cycle arrest, on the one hand, implies that the core taxane ring may play a critical role in inducing cell death and, on the other hand, suggests that paclitaxel might induce apoptosis from other phases of the cell cycle by a similar mechanism.

Alkaloids↗
PubMed1999

Diphtheria toxin fused to granulocyte-macrophage colony-stimulating factor and Ara-C exert synergistic toxicity against human AML HL-60 cells.

Human granulocyte-macrophage colony-stimulating factor fused to truncated diphtheria toxin (DT388-GM-CSF) sensitized wild-type and Bcl2-overexpressing HL60 human leukemia cells to intoxication by Ara-C based on proliferation and clonogenic assays. The toxin/drug combination showed dramatic synergistic toxicity with combination indices of < 0.1. Synergy was not seen with two other protein synthesis inhibiting drugs--ricin and cycloheximide nor with GMCSF alone. No changes in Ara-C incorporation into cellular DNA or cell cycle occupancy were seen. As compared to exposure to DT388-GM-CSF or Ara-C alone, co-treatment produced significant increases in cytosolic accumulation of cytochrome c, a higher percentage of cells with loss of mitochondrial membrane potential and an increase in reactive oxygen species and morphologic changes of apoptosis, and a greater induction of poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor 45 (DFF45) cleavage activities of caspase 3. Co-treatment did not significantly alter Bcl2, Bcl-xL, Bax or Fas receptor (FasR), but modestly increased Fas ligand (FasL) protein. These finding suggest that co-treatment with DT388-GM-CSF may lead to a lowered apoptotic threshold and clonogenic survival of human AML blasts due to Ara-C. These observations also suggest that clinical trials of combination therapy may be warranted in patients with AML.

Acute Disease↗
PubMed1999

DT388-GM-CSF, a novel fusion toxin consisting of a truncated diphtheria toxin fused to human granulocyte-macrophage colony-stimulating factor, prolongs host survival in a SCID mouse model of acute myeloid leukemia.

Despite significant advances in the treatment of acute myeloid leukemia (AML), the majority of patients will succumb to drug-resistant AML. To overcome this resistance, we have developed a novel fusion toxin consisting of the catalytic and translocation subunits of diphtheria toxin (DT388) linked to human granulocyte-macrophage colony-stimulating factor (GM-CSF). In vitro, DT388-GM-CSF demonstrated significant activity against numerous AML cell lines and fresh AML blasts. To determine its in vivo efficacy, we developed an in vivo model of human AML in severe combined immunodeficiency (SCID) mice injected intravenously with 1 x 10(7) HL-60 cells (AML-M2 cell line). The SCID mice developed abdominal masses, infiltration of the liver and bone marrow, and peripheral blasts with a median survival of 42.5 days. We tested DT388-GM-CSF, ara-C, human GM-CSF, and DAB389IL-2, which were injected intraperitoneally on days 2-6 in this model. DT3-GM-CSF significantly improved survival of the SCID mice over Ara-C, DAB389IL-2, or control (P < 0.001). DT388-GM-CSF-treated mice who developed leukemia exhibited no difference in the number of GM-CSF receptors (P = 0.39), ligand affinity (P = 0.77), or sensitivity (P = 0.56) to DT388-GM-CSF as compared to the controls. Frank leukemia in DT388-GM-CSF-treated mice may be due to incomplete penetration of drug into tissues rather than cellular resistance. DT388-GM-CSF is an active therapeutic agent in our SCID mouse model of AML with a unique mechanism of action and differing toxicities than current cytotoxic agents.

Animals↗
PubMed1999

Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells.

This study was undertaken to investigate the influence of the peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on the proliferation, apoptosis and tumorigenesis of breast cancer cells. PPARgamma investigation has been largely restricted to adipose tissue, where it plays a key role in differentiation, but recent data reveal that PPARgamma is expressed in several transformed cells. However, the function of PPARgamma activation in neoplastic cells is unclear. Activation of PPARgamma with the known prostanoid agonist 15-deoxy-Delta12,14-prostaglandin J(2) (15dPGJ(2)) or the thiazolidinedione (TZD) agonist troglitazone (TGZ) attenuated cellular proliferation of the estrogen receptor-negative breast cancer cell line MDA-MB-231, as well as the estrogen receptor-positive breast cancer cell line MCF-7. This was marked by a decrease in total cell number and by an inhibition of cell cycle progression. Addition of 15dPGJ(2) was not associated with an increase in cellular differentiation, as has been seen in other neoplastic cells, but rather induction of cellular events associated with programmed cell death, apoptosis. Video time-lapse microscopy revealed that 15dPGJ(2) induced morphological changes associated with apoptosis, including cellular rounding, blebbing, the production of echinoid spikes, blistering and cell lysis. In contrast, TGZ caused only a modest induction of apoptosis. These results were verified by histochemistry using the specific DNA stain DAPI to observe nuclear condensation, a marker of apoptosis. Finally, a brief exposure of MDA-MB-231 cells to 15dPGJ(2) initiated an irreversible apoptotic pathway that inhibited the growth of tumors in a nude mouse model. These findings illustrate that induction of apoptosis may be the primary biological response resulting from PPARgamma activation in some breast cancer cells and further suggests a potential role for PPARgamma ligands for the treatment of breast cancer.

Animals↗
PubMed1999

Mitotic chromosomal bcl-2. I. Stable expression throughout the cell cycle and association with isolated chromosomes.

Bcl-2 is present in a cytoplasmic distribution in cells that express high levels of this oncoprotein. In contrast, using immunocytochemistry in cells expressing low levels of bcl-2, such as KB human carcinoma cells, we and others have shown that bcl-2 is present on the surface of mitotic chromosomes. However, monoclonal antibodies reactive with an epitope representing amino acids 41-54 of the bcl-2 sequence did not detect bcl-2 in other phases of the cell cycle. This study extended those earlier findings to determine if bcl-2 was expressed as a cyclin or if this pattern was an artifact of immunocytochemistry. Immunofluorescence studies in several other human cell lines showed the same mitotic distribution of bcl-2. Other studies using flow cytometry also showed selective mitotic phase detection of bcl-2. A comparison of available commercial antibodies showed that, in spite of reactivity with denatured bcl-2 on Western blots, clear reactivity with bcl-2 in fixed cells was found only with those reactive with the (a.a. 41-54) epitope. With RNase protection and Western blot analyses, cells synchronized at various stages of the cell cycle showed constant levels of bcl-2 mRNA and protein. Analysis of bcl-2 using Western blots showed a band with the same apparent molecular weight as that seen in comparison with authentic bcl-2 overexpressed in the cytoplasm. The retention of bcl-2 on chromosomes in unfixed, permeabilized preparations was influenced by protease treatment, phosphate, and pH. Studies using isolated chromosomes showed that much of the bcl-2 in these cells was attached to chromosomes in mitosis, had the expected molecular weight, and was phosphorylated in the same manner as that seen in whole-cell extracts. These results show that bcl-2 is not a cyclin and that the bcl-2 localized on chromosomes is the same molecule seen by immunoblotting. These results suggest that the reactive (a.a. 41-54) epitope present in bcl-2 is somehow modified or masked in interphase.

Animals↗
PubMed1999

Mitotic chromosomal bcl-2. II. Localization to interphase nuclei.

We have previously shown, by immunofluorescence of fixed cells, that bcl-2 is found only in mitotic chromosomes in KB cultured human tumor cells expressing low levels of this oncoprotein. However, other studies showed that bcl-2 did not change its levels during the cell cycle when analyzed using Western blots. In this study we analyzed the distribution of bcl-2 during interphase, the point at which it is undetectable by immunofluorescence, using biochemical extraction, immunoprecipitation, and cell fractionation with Western blots. Interestingly, when carefully examined by immunofluorescence in fixed cells, the earliest point in the cell cycle showing bcl-2 localization was early G2, in which bcl-2 could be found within the intact nucleus. In spite of showing no immunofluorescence reaction in fixed interphase cells, immunoprecipitation of gentle detergent extracts showed that bcl-2 from interphase cells reacted readily with the antibody used (#124) after extraction. However, immunoprecipitation using anti-bcl-2 followed by Western blots using anti-Bax showed that, unlike overexpressing cells, this bcl-2 was not complexed with Bax. Classical cell fractionation methods were used to separate nuclei from cytosol and cell membranes. Surprisingly, these experiments clearly showed that essentially all of the bcl-2 in interphase KB cells was present in the nucleus. Therefore, the lack of reaction in fixed cells with anti-bcl-2 antibody reflects either a masking or a conformational change of the reactive epitope in bcl-2 present within the nucleus. By correlation, this change may be related to the phosphorylation of bcl-2 that occurs just before mitosis. The nature of this novel yet highly conserved nuclear form of bcl-2 and the understanding of its function will require further study.

Blotting, Western↗
PubMed1999

Cytochemical methods for the detection of apoptosis.

Detection of apoptotic cell death in cells and tissues has become of paramount importance in many fields of modern biology, including studies of embryonic development, degenerative disease, and cancer biology. In addition to methods that employ biochemical analysis of large populations of cells, cytochemical methods have recently been extensively used both in individual cells and in tissues. Most of these methods exploit properties of dying cells that are more or less specific for the apoptotic process. However, considerable confusion exists over the interpretation of some of these methods and their usefulness in all settings. This review attempts to summarize the more recent advances in cytochemical detection of apoptosis and emphasizes some of the pitfalls that confuse the interpretation of results of these methods.

Apoptosis↗
PubMed1999

Hepatotoxicity in cancer patients receiving erb-38, a recombinant immunotoxin that targets the erbB2 receptor.

To exploit overexpression of erbB2 in human cancers, we constructed a single-chain immunotoxin (erb-38) that contains the Fv portion of monoclonal antibody e23 fused to a truncated form of Pseudomonas exotoxin A. In a Phase I study, five breast cancer patients and one esophageal cancer patient received three doses of erb-38 at 1.0 and 2.0 microg/kg. Hepatotoxicity was observed in all patients. Immunohistochemistry showed the presence of erbB2 on hepatocytes explaining the liver toxicity of erb-38. We suggest that targeting of tumors with antibodies to erbB2 armed with radioisotopes or other toxic agents may result in unexpected organ toxicities due to erbB2 on normal cells.

ADP Ribose Transferases↗
PubMed1998

Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L.

Cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by malignantly transformed cells. However, the reason for secretion of this man 6-phosphate-containing lysosomal protease into the extracellular medium is not clear. We wished to determine whether there is a region within the primary sequence of the proenzyme form of cathepsin L which affects its subcellular and extracellular localization. High-level transient expression of human procathepsin L in mouse NIH 3T3 cells results in the secretion of most of this protein into the extracellular medium. At the same time, the endogenous mouse procathepsin L in these nontransformed cells is found in its usual location in lysosomes. Mutants of human procathepsin L with carboxy-terminus deletions involving the last 11 amino acids are not secreted into the medium. Deletion of as little as two amino acids, Thr and Val, from the carboxy terminus, blocked the secretion of the protein but did not affect its enzyme activity, posttranslational processing, or subcellular distribution. Replacement of Thr-Val by two bulky amino acids Tyr-Asn allowed secretion of the procathepsin L, but the replacement of these two amino acids by nonbulky alanines prevented its secretion. Single alanine substitutions of the last six amino acids (ASYPTV) indicated that substitution by alanine of Y or T does not affect the secretion of hproCAT L, but alanine substitutions of S, P, or V completely blocked its secretion into the culture medium. We therefore conclude that the carboxy terminus of procathepsin L contains a sequence essential for its secretion.

3T3 Cells↗