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M E Stearns

Publications and source records attributed to M E Stearns.

At least 55 records · Page 3Linked to original sources
PubMed1991

Isolation and characterization of PC-3 human prostatic tumor sublines which preferentially metastasize to select organs in S.C.I.D. mice.

We have developed and partially characterized a mouse model system for studying human prostate tumor cell metastases in vivo. To develop this model we have selected highly invasive (3 x I.) and non-invasive (3 x N.I.) PC-3 human prostatic tumor sublines based on enhanced and diminished capacities to migrate across a reconstituted basement membrane barrier (Matrigel) in Boyden chamber chemotactic assays. When the 3 x I. cells were injected intravenously (i.v.) in the tail vein of severe combined immune deficient (scid) mice, the cells initially metastasized to a wide variety of tissues as demonstrated by using [125I] IUdR labeled cells and histology. Four distinct sublines were eventually isolated which preferentially metastasized at approximately 80% efficiency to the lumbar vertebrae (PC-3 ML), the mandibular region of the right cheek (PC-3 MC), the rib cartilage (PC-3 MR), and the right front knee bone (PC-3 MK), respectively. Implantation experiments at different sites indicated that organ metastases may somehow be conferred on the tumor subclones by the host tissue.

Animals↗
PubMed1991

Evidence that estramustine binds MAP-1A to inhibit type IV collagenase secretion.

Estramustine is a novel anti-microtubule drug shown to bind MAP-1 and MAP-2 (microtubule-associated proteins) in vitro. In this paper we have shown that estramustine specifically binds MAP-1A in Du 145a cells, resulting in disruption of MAP-1A microtubules and inhibition of type IV collagenase secretion. Immunofluorescence studies revealed that at 30 microM levels estramustine blocked type IV collagenase secretion by partial disruption of the MAP-1A microtubule networks. Immunoprecipitation studies with polyclonal antibodies provided quantitative evidence that 30-60 microM estramustine blocked secretion of a 105 x 10(3) Mr type IV collagenase. Pulse-labeling experiments confirmed that the effect was not a result of inhibition of either protein synthesis or altered rates of type IV collagenase turnover. Finally, drug uptake studies with [3H]estramustine, scintillation counting and fluorography demonstrated that the principal target of the drug was MAP-1A. For the first time we have shown that the drug blocks secretion by binding MAP-1A and causing incomplete disruption of the microtubule networks.

Cell Line↗
PubMed1990

Properties of kinesin isolated from human prostatic DU 145 tumor cells and bovine brain.

We have isolated and compared the 116-kilodalton (kDa) kinesin heavy chain from DU 145 human prostatic tumor cells and bovine brain. Comparative sodium dodecyl sulfate - polyacrylamide gel electrophoreses (SDS-PAGE), Western blots, and proteolytic digestion analysis all showed that the 116-kDa polypeptides from both sources were indistinguishable. Polyclonal antibodies raised against sea urchin kinesin cross-reacted with both brain and DU 145 kinesin on Western blots. SDS-PAGE and A-5m chromatographic studies indicated that kinesin forms a quarternary heteropolymer of approximately 400 kDa. DU 145 cells had three proteins of 116, 72, and 64 kDa forming the heteropolymer, in a 2:1:1 ratio, whereas brain cells appeared to have equimolar amounts of the 116-kDa heavy chain and a 64-kDa light chain.

Adenosine Triphosphatases↗
PubMed1989

Deprivation/enhancement fantasy patterns in children.

Previous studies have shown that females tell stories that change from negative experiences (deprivation) to positive experiences (enhancement) and males tell stories that change in the opposite direction. The current study examines this phenomenon in children using a different story-telling procedure. Results were not consistent with those of previous studies.

Child↗
PubMed1988

Blocking of collagenase secretion by estramustine during in vitro tumor cell invasion.

The effects of the antitumorigenic drug estramustine on tumor cell membrane penetration (invasion) were investigated in vitro by utilizing a synthetic basement membrane system (a modified Boyden chamber). Tumor cells were plated on a "partition barrier," consisting of a porous filter (8-micron pores) which was coated with a reconstituted basement membrane matrix (Matrigel), and induced to migrate across the barrier with conditioned medium obtained from 9DU 145 human prostatic tumor cells (passage 9). Quantitative radiolabeling studies demonstrated that specially isolated lines (isolated by several passages through the Matrigel) of DU 145 cells, A2058 melanoma, and B16-F10 melanoma cells were highly invasive such that 15 to 20% migrated across a 1-mm-thick Matrigel layer within 5 h at 37 degrees C. NIH-3T3 cells, mouse fibroblasts, and 20DU 145 cells (passage 20) exhibited little or no membrane invasive behavior. Micromolar concentrations of estramustine (30 to 120 microM) inhibited invasion by the invasive cell lines in a dosage-dependent fashion. Quantitative enzymatic assays and radioimmune assays demonstrated that estramustine inhibited membrane invasion by blocking type IV collagenase secretion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots confirmed that 30 to 60 microM estramustine blocked secretion of a Mr 105,000 collagenase protein. Indirect studies showed that a collagenase antibody raised against the Mr 105,000 protein and inhibitors of proteinase activity, including a metalloproteinase inhibitor, and 1,10-phenanthroline, blocked invasion. Because the antibodies inhibited type IV collagenase digestion of 3H-mouse type IV collagen, and invasion simultaneously, it is proposed that collagenolytic activity is involved in invasion. These data demonstrate that estramustine blocks proteinase secretion, and suggest that estramustine may be a useful therapeutic drug for the prevention of metastasis.

Cell Division↗
PubMed1988

Estramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells.

The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly. Turbidimetric (A350), SDS-PAGE, and electron microscopic studies revealed that micromolar estramustine inhibited assembly promoted by the 330-kD protein. Similarly, estramustine inhibited binding of the 330-kD protein to 6-S microtubules independently stimulated to assemble with taxol. Immunofluorescent studies with beta-tubulin antibody (27B) and MAP-1 antibody (MI-AI) revealed that 60 microM estramustine (a) caused disassembly of MAP-1 microtubules in DU 145 cells and (b) removed MAP-1 from the surfaces of microtubules stabilized with 0.1 microM taxol. Taken together the data suggested that estramustine binds to a 330-kD MAP-1-like protein to disrupt microtubules in tumor cells.

Blotting, Western↗
PubMed1988

Estramustine binds MAP-2 to inhibit microtubule assembly in vitro.

We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine's relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2. Calculations from kinetic parameters and non-linear regression analysis gave a Kd of 15 microM, and a Bmax of 3.4 x 10(-7)M ml-1. Extrapolation of this value suggested that each MAP-2 molecule binds approximately 20 molecules of estramustine. Microtubule assembly studies and SDS-polyacrylamide gel electrophoresis revealed that at 20-60 microM levels, estramustine inhibited the association of MAPs with taxol microtubules. Turbidity (A350) studies further demonstrated that 20-60 microM-estramustine inhibited MAP-2-driven tubulin assembly and produced microtubule disassembly. Electron-microscopic studies confirmed the centrifugation and turbidity results. The data demonstrated that estramustine can bind MAPs and MAP-2 specifically, thereby inhibiting microtubule assembly.

Animals↗
PubMed1987

Evidence that MAP-2 may be involved in pigment granule transport in squirrel fish erythrophores.

We have demonstrated the presence of MAP-2 in squirrel fish erythrophores using SDS-PAGE, immunoblot, and immunoprecipitation techniques. The monoclonal antibodies used (AP-9, -13, -14) were raised against distinct antigenic sites on Chinese hamster brain MAP-2. Immunoprecipitation studies demonstrated that all three antibodies bind a 300 K protein found in crude cell extracts and in partially purified MAP fractions isolated from erythrophores of the squirrel fish Holocentrus rufus. Immunofluorescent studies confirmed that the 300 K protein was present in cultured erythrophores. Studies of cells induced to aggregate and disperse their pigment granules revealed that the 300 K protein comigrated with the pigment, suggesting that the 300 K protein may constitute part of the "alpha-cytomatrix" involved in pigment translocations.

Animals↗
PubMed1987

Hormone-independent, non-alkylating mechanism of cytotoxicity for estramustine.

Over two decades, experience with estramustine has provided limited data which support an estrogenic mechanism of action and no data which indicate the nitrogen mustard involvement in the cytotoxic properties of the drug. Consideration of the carbamate-ester portion of estramustine supports the pharmacokinetic evidence that estramustine has a long half life since enzymatic hydrolysis of the carbamate is an uncommon event. Using a variety of immunocytochemical and cellular morphology procedures, estramustine per se has been found to express anti-cytoskeletal properties through non-covalent binding to microtubule associated proteins (MAP's). In both fish erythrophores and in dividing human prostatic carcinoma cells, estramustine exerts an antimicrotubule effect at micromolar concentrations. Thus, estramustine possesses unique pharmacology and protein binding specificity. As such, it should not be classified as an alkylating agent. The estrogenic effects, while possibly of relevance to clinical administration, are not the primary mechanism by which the drug exerts cytotoxicity.

Alkylation↗
PubMed1987

Evidence for intermediate filaments in squirrelfish erythrophores of Holocentrus ascensionus (Rufus).

We have documented the presence of intermediate filaments (IF) in cultured erythrophores of the squirrelfish Holocentrus ascensionus (Rufus). SDS-PAGE and Western blots with monoclonal antibodies T11 and R12 demonstrated that isolated IF consisted of a pair of polypeptides of 54 and 52 kDa. Immunofluorescent studies revealed that the two proteins formed prominent radially oriented IF networks in erythrophores. Immunoelectron microscopic studies showed that the IF were distributed in a "spider-web"-like network of filaments which occasionally intersected with the microtubule surfaces. The IF proteins also were found in fish iridiphores but not in fish epithelial cells which cocultured with the chromatophores.

Animals↗
PubMed1987

Plasmodium falciparum: fine structural changes in the cytoskeletons of infected erythrocytes.

Following parasitization by Plasmodium falciparum, numerous changes take place in the host erythrocyte membrane. In this study, we used the technique of whole cell mount electron microscopy to determine if the ultrastructure of the erythrocyte cytoskeleton changed following parasitization with knobby and knobless strains of P. falciparum. Using this technique, a network of spectrin filaments (3-10 X 45-120 nm) branching from electron dense junctions (15-25 nm in diameter), the presumed site of bands 4.1 and actin, were visualized. The overall architecture of normal and parasitized erythrocyte cytoskeletons was the same: however, additional patches (35 to 60 nm in size) and aggregates (30 X 150 nm) of electron dense material were present in parasitized skeletons. The ultrastructure of knobby and knobless cytoskeletons was similar, except knobless skeletons usually did not possess the larger aggregates of material. Antigens associated with the erythrocyte cytoskeleton of cells infected with knobby and knobless strains, but not uninfected cells, were demonstrated by indirect immunofluorescence. Results suggest that antigens, associated with the erythrocyte cytoskeleton, may contribute to perturbations in the host erythrocyte membrane.

Animals↗
PubMed1987

Localization of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs.

Plasmodium falciparum parasites that induce knobs in the host erythrocyte membrane (K+ phenotype) synthesize a 90 kDa histidine-rich protein (PfHRP-1), whereas knobless variants do not. A monoclonal antibody (mAb 89) to PfHRP-1, in combination with cryo-thin section immunoelectron microscopy, localized the antigen in the parasitophorous vacuolar space and vesicles within the erythrocyte cytosol. Additional immunoelectron microscopic studies showed that PfHRP-1 was also associated with submembranous electron-dense material under knobs and with microfilaments of the host erythrocyte skeletal network. Immunofluorescence and immunoelectron microscopy of intact, non-fixed K+ infected erythrocytes using mAb 89 and a rabbit antiserum raised against purified PfHRP-1, failed to identify any surface exposed epitopes. These antibodies also failed to block cytoadherence of infected erythrocytes to C32 melanoma cells or to affect macrophage phagocytosis of infected erythrocytes.

Animals↗
PubMed1987

Polarized pigment granule transport occurs in the absence of microtubules in squirrelfish erythrophores: studies of the effects of estramustine.

We have re-examined the involvement of microtubules in the process of pigment granule transport in squirrelfish erythrophores in situ (i.e. on scales). Light-microscopic studies revealed that following exposure to 5 microM-nocodazole for 1 h at 4 degrees C erythrophores retained an ability to aggregate and disperse their pigment uniformly, though at reduced rates. Serial thick-section stereo high-voltage electron-microscopic studies showed that the entire microtubule population was removed by drug treatment and that the microtubules were not reassembled as a result of pigment translocation processes in the presence of reduced levels of nocodazole (0.4 microM). Immunofluorescence microscopic studies confirmed that nocodazole (0.5-1 microM) produced rapid disassembly of the microtubules. Whole-mount electron-microscopic studies showed that the pigment granules were suspended in a cross-linking network of 3-10 nm filaments, which appeared to support ordered pigment transport in situ in the absence of microtubules. Drug inhibition studies showed that micromolar levels of estramustine, a novel anti-MAPs (microtubule-associated proteins) drug, reversibly inhibited pigment transport. The results suggest that an estramustine-sensitive cytomatrix component might produce polarized pigment transport in intact erythrophores.

Animals↗
PubMed1986

The cytomatrix regulates "resolute" transport in erythrophores.

Light microscopic studies have indicated that most microtubule-directed transport is either saltatory or resolute in nature. The latter form of transport is an intriguing phenomenon, because it commonly involves the unidirectional bulk motion of an organelle(s) such as chromosomes in dividing cells or pigment granules in chromatophores. We have investigated the ultrastructural and biochemical basis for the resolute transport of pigment in chromatophores. Light and EM studies of erythrophores in situ have clearly shown that when the microtubules were completely removed with nocodazole, resolute transport continued and was stimulated by aggregating and dispersing agents. Light and electron microscopic studies of cultured erythrophores permeabilized with digitonin indicated that resolute motion was produced by a cytomatrix of 3 to 7 nm filaments. Immunofluorescent analysis with several monoclonal antibodies raised against MAP-2 further demonstrated that MAP-2 was an important component of the contractile cytomatrix that powers pigment aggregation and dispersion. We conclude that a microtubule-associated cytomatrix normally produces resolute pigment transport in chromatophores.

Animals↗
PubMed1986

Relationship of glutathione depletion and inhibition of glutathione-S-transferase activity to the antimitotic properties of estramustine.

A depletion of intracellular glutathione (GSH) is accompanied by a subsequent inhibition of GSH-S-transferase activity in a DU145 human prostatic carcinoma cell line treated with cytotoxic concentrations of estramustine. When GSH depletion reached approximately 50% of normal (approximately 2 hours after 10 microM estramustine), the mitotic index of logarithmically dividing cultures began to increase. A linear increase from 3.14% to 22.5% occurred during the period of 2-24 hours following 10 microM estramustine. Morphological studies showed that mitotic figures accumulated in metaphase and had abnormalities consistent with spindle malformation. Nocodazole and cytochalasin B also possess anticytoskeletal properties, but had little effect upon the intracellular levels of GSH or its associated transferase enzymes. The constituent moieties of estramustine, estradiol, and nor-mechlorethamine had effects on thiol metabolism which were dissimilar from estramustine, confirming previous findings that the unmetabolized parent drug is responsible for pharmacological activity. Estramustine had no effect upon GSH reductase activity, suggesting that drug toxicity was not a general thiol phenomenon. Buthionine sulfoximine decreased intracellular GSH in DU145 cells and enhanced the cytotoxic potential of estramustine in this cell line. The anticytoskeletal and antimitotic properties of estramustine may be enhanced by the drug-induced GSH imbalance and subsequent effects on GSH-S-transferase enzymes.

Buthionine Sulfoximine↗