Biomedical subjects

M J Weber

Publications and source records attributed to M J Weber.

At least 127 records · Page 7Linked to original sources

PubMed1986

Tyrosine phosphorylation in cells treated with transforming growth factor-beta.

Cells stimulated with epidermal growth factor (EGF) or any one of a diverse group of other mitogenic agents display an increased tyrosine phosphorylation of a pair of 42,000 Mr proteins. Transforming Growth Factor-beta (TGF-beta) is able to potentiate the mitogenic effects of Epidermal Growth Factor on some fibroblastic cells (such as the NRK-49F cell line) and, in addition, permits the anchorage-independent growth of these cells. In this study we asked whether these growth-regulatory actions of Transforming Growth Factor-beta are associated with changes in tyrosine phosphorylation of cellular proteins, in particular the 42,000 Mr proteins. We found no effect of Transforming Growth Factor-beta on the extent or time-course of tyrosine phosphorylation, either by itself or in combination with Epidermal Growth Factor. Since the tyrosine phosphorylation of the 42,000 Mr proteins is stimulated both by receptors with tyrosine kinase activity and by diacylglycerol analogs (but not by Transforming Growth Factor-beta), we suggest that the activity of the receptor for Transforming Growth Factor-beta is linked neither to tyrosine phosphorylation nor to phosphatidyl inositol turnover.

Animals↗

PubMed1986

Cellular localization of the molecular forms of acetylcholinesterase in primary cultures of rat sympathetic neurons and analysis of the secreted enzyme.

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.

Acetylcholinesterase↗

PubMed1985

Molecular properties of a cholinergic differentiation factor from muscle-conditioned medium.

Conditioned medium by a variety of rat non-neuronal cells contains a protein involved in the differentiation of cholinergic neurons in cultures prepared from newborn rat superior cervical ganglion, from nodose ganglion, and from embryonic spinal cord. We have determined some hydrodynamic properties of this factor using as a bioassay the increase in choline acetyltransferase activity in sympathetic neurons grown for 12-15 days in the presence of the factor. The Stokes' radius, measured by molecular sieving chromatography on an Ultrogel AcA 44 column, was similar to that of ovalbumin (27.6 A). By analysis on 5-20% linear sucrose gradients made in H2O and D2O, we determined the partial specific volume (0.68 ml X g-1 and the sedimentation coefficient (2.1S). These data allowed the calculation of the molecular weight (21,000) and the frictional ratio f/fo (1.56). The elution pattern of the factor from a SynChropak CM 300 HPLC cation exchange column suggested that it was a basic protein. The activity of this factor was unaffected by heat treatment at 100 degrees C for 10 min.

Animals↗

PubMed1985

Protein phosphorylation in a tetradecanoyl phorbol acetate-nonproliferative variant of 3T3 cells.

The 3T3-TNR9 cell line is a variant of Swiss 3T3 cells which does not respond mitogenically to tumor promoters, but does respond mitogenically to epidermal growth factor, fibroblast growth factor, and serum. To elucidate differences between tumor promoters and polypeptide mitogens in the pathway(s) of mitogenesis which might be responsible for the nonresponsiveness of the 3T3-TNR9 cells, we have examined in these cells the early protein phosphorylation events known to be associated with mitogenesis in the parental 3T3 cells. We find that the 3T3-TNR9 cells display levels of tetradecanoyl phorbol acetate binding and of a calcium- and phospholipid-dependent protein kinase activity which are at least the equal of those seen in the parental 3T3 cells, implicating some postreceptor event in the nonmitogenic phenotype. In addition, we find that phosphorylation of the epidermal growth factor receptor and of 80-kDa and 22-kDa proteins, as well as the tyrosine phosphorylation of a 42-kDa protein, all proceed normally in the nonmitogenic variant, even though these phosphorylations must depend on the activation of different kinases. Thus, all these early phosphorylation reactions are intact in the 3T3-TNR9 cells. Although these phosphorylations may be necessary, they clearly are insufficient to trigger mitogenesis.

Animals↗

PubMed1985

Treatment of systemic candidiasis in neutropenic dogs with ketoconazole.

The present study evaluated the activity of ketoconazole in neutropenic dogs with systemic candidiasis. Five dog pairs were made neutropenic by intravenous cyclophosphamide (50 mg/kg) and challenged with either 10(6) or 10(7) colony-forming units (CFU) of Candida albicans. Half of the dogs received ketoconazole (10 mg/kg) daily beginning 24 h after challenge. All were killed at 96 h and liver, spleen, and kidney were cultured. Of four dogs given 10(6) CFU, two untreated dogs had 9 X 10(3) to 1 X 10(5) CFU/g wet tissue, compared to 0 CFU in ketoconazole-treated dogs. With inoculum increased to 10(7) CFU, three untreated dogs had 2 X 10(4) to 3 X 10(5) CFU/g wet tissue, while three ketoconazole dogs had 0-5 X 10(3) CFU/g wet tissue. The effect of ketoconazole on autologous marrow reconstitution in dogs with systemic candidiasis was examined by infusing autologous cryopreserved marrow into four dogs one day after lethal whole body irradiation (800 rad). Once neutropenic, they were challenged with 10(7) CFU of C. albicans. Two dogs received no ketoconazole and died of disseminated candidiasis, without marrow reconstitution. Two dogs received ketoconazole for 25 days. Prompt marrow recovery occurred and they remained healthy. There was no evidence of infection at death. These studies quantitatively demonstrate the in vivo effectiveness of ketoconazole in reducing tissue infection with C. albicans in neutropenic dogs. They provide in vivo evidence that ketoconazole can prevent or cure systemic candidiasis in the bone marrow transplant setting without significant inhibition of marrow recovery.

Agranulocytosis↗

PubMed1984

Calcium interactions in pulmonary surfactant.

The surfactant properties of natural bovine pulmonary surfactant, its lipid extracts and acetone precipitates of lipid extracts have been examined with an artificial alveolus model, the pulsating-bubble surfactometer. At bulk concentrations of 0.4% (wt./vol.) phospholipid in saline, all three preparations exhibited surfactant activity, i.e., were capable of reducing the surface tension of the pulsating bubble to approx. 27 dynes/cm at maximum bubble radius and to near zero at minimum bubble radius. At a concentration of 0.1% (wt./vol.) in saline, only natural surfactant was effective. Acetone-precipitated surfactant at 0.1% (wt./vol.) achieved these criteria in the presence of 5 mM calcium, but 15-20 mM calcium was required to restore the surfactant activity of lipid extract surfactant. Chemical analysis revealed that lipid extraction decreases the protein content but does not alter the endogenous calcium levels. A calcium requirement for natural surfactant could only be demonstrated after repeated treatment with chelators for divalent cations. Surfactant activity was restored by low levels of calcium or high levels of magnesium. Paradoxically, a calcium requirement could not be demonstrated by treating acetone-precipitated lipid extract with chelators. The subtle differences noted between natural, lipid extract and acetone-precipitated lipid extract surfactant with the pulsating-bubble assay show that the latter preparations do not represent simplified model systems for the natural product.

Acetone↗

PubMed1984

Molecular events leading to enhanced glucose transport in Rous sarcoma virus-transformed cells.

Transformation by Rous sarcoma virus results in a dramatic increase in the rate at which the transformed cells transport glucose across the cell membrane. The increased transport rate is a consequence of an increased number of transporters in the transformed cells. Utilizing antibody raised against the purified human erythrocyte glucose transporter, we have identified the glucose transporter as a membrane glycoprotein with a monomer Mr of approximately 41,000. The increased rate of glucose transport is dependent on the activity of pp60src, the transforming protein of Rous sarcoma virus. This protein has been shown to be a protein kinase that phosphorylates on tyrosine residues. We have examined the tyrosine phosphorylation of a major cellular protein of Mr 36,000 in cells infected with a panel of partially transforming mutants of Rous sarcoma virus. One of these mutants (CU2) increases the rate of glucose transport only slightly and does not render the infected cells fully anchorage independent or tumorigenic (although other transformation parameters are fully induced). Cells infected with this mutant display a 36,000-dalton protein that is phosphorylated to a considerably lesser extent than cells infected with wild-type virus. Analyses of this sort may help to identify the cellular targets of pp60src whose phosphorylation is necessary for the increased glucose transport rate.

3-O-Methylglucose↗

PubMed1984

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. II. Regulation of 16 S acetylcholinesterase by conditioned medium.

The molecular forms of acetylcholinesterase (AcChE) have been studied in primary cultures of newborn rat sympathetic neurons grown either in the absence (CM- cultures) or in the presence (CM+ cultures) of muscle conditioned medium. The cultures were treated with a mitotic poison to eliminate non-neuronal cells. CAT activity increased with time in culture 4- to 20-fold faster in CM+ than in CM- cultures. In agreement with previous experiments (J.P. Swerts, A. Le Van Thaï, A. Vigny, and M.J. Weber, 1983, Develop. Biol. 100, 1-11), AcChE activity developed at a 3-fold lower rate in CM+ than in CM- cultures. This deficit in AcChE activity in CM+ cultures resulted from a deficit in the number of enzyme molecules immunoprecipitable with an antiserum raised against rat brain AcChE. In both types of cultures, AcChE forms were separated by sucrose gradient sedimentation into three main peaks corresponding to the 16 S and 10 S forms and a mixture of the 6.5 and 4 S forms. In 3-day-old CM+ and CM- cultures, the 16 S form represented 2% of the total activity. After 12-26 days, the percentage of 16 S form raised to 15-30% in CM- cultures, but remained lower than 5% in CM+ cultures. This difference was also observed when AcChE molecular forms were analyzed in the presence of protease inhibitors. A similar result was obtained by comparing cultures grown with and without a macromolecular factor partially purified from conditioned medium. These results suggest that an inverse relationship exists between the presence of 16 S AcChE and the presence of cholinergic synapses in these cultures.

Acetylcholinesterase↗

PubMed1984

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. III. Effects of sodium butyrate.

The effects of Na butyrate on the differentiation of newborn rat sympathetic neurons in primary cultures have been studied. Butyrate did not affect the long-term survival of these neurons in the presence of optimal concentrations of nerve growth factor, but decreased in a dose-dependent manner their protein content. In the range, 0.5-20 mM, butyrate did not modify the specific activity of lactate dehydrogenase in these cultures. Choline acetyltransferase activity developed at a 4.5- to 12-fold higher rate in cultures grown with 1-5 mM butyrate than in its absence. Concomitantly, tyrosine hydroxylase, dopa decarboxylase, dopamine-beta-hydroxylase, and acetylcholinesterase were depressed in cultures grown with butyrate. The deficit in acetylcholinesterase total activity was accompanied by an inhibition of the development of the asymmetric 16 S form of the enzyme. The deficit in tyrosine hydroxylase activity did not result from either a modification of the app Km for the enzyme's cofactor or a modification of its state of cAMP-dependent phosphorylation, but from a decrease in the number of immunoprecipitable enzyme molecules. A similar result was obtained with acetylcholinesterase. Butyrate thus reproduced in a qualitative manner the effects of a macromolecular factor purified from muscle conditioned medium on these neurons (J. P. Swerts, A. LeVan Thaï, A. Vigny, and M. J. Weber (1983) Dev. Biol. 100, 1-11; J. P. Swerts, Le Van Thai, and M. J. Weber (1984) 103, 230-234), raising the hypothesis of a common pathway in the regulation of neurotransmitter phenotype by these two agents.

Acetylcholinesterase↗

PubMed1984

Acetylcholine metabolism by cultured neurons from rat nodose ganglia: regulation by a macromolecule from muscle-conditioned medium.

Acetylcholine metabolism has been studied in primary cultures of neurons dissociated from newborn rat nodose ganglia. Neither nerve growth factor nor muscle-conditioned medium had any detectable effect on the long-term survival of these neurons, which appeared well differentiated upon phase-contrast and electron microscopic examination. [3H]Acetylcholine synthesis and accumulation by 2-3-week old nodose cultures and choline acetyltransferase activity were increased (2.0 +/- 0.15)-fold and (2.0 +/- 0.48)-fold, respectively, upon growth with muscle-conditioned medium, whereas acetylcholinesterase was decreased (1.5 +/- 0.1)-fold (means +/- SEM, n = 5-9). The same effects were observed when comparing nodose cultures grown in the presence of proteins purified from conditioned medium in four fractionation steps. This purified material had no effects on the protein content of the cultures, or on lactate dehydrogenase activity, and thus did not affect the overall growth of the cultures. We demonstrate that this factor copurifies with a factor(s) involved in the regulation of choline acetyltransferase and acetylcholinesterase in neuron cultures from newborn rat superior cervical ganglia [Swerts, Le Van Thai, Vigny and Weber (1983) Devl Biol. 100, 1-11] and from embryonic day 13 rat embryo spinal cord [Giess and Weber (1984) J. Neurosci. 4, 1442-1452]. Although the cholinergic factor from muscle-conditioned medium has not been purified to homogeneity, the data suggest that the same extracellular, macromolecular factor may be involved in the regulation of acetylcholine metabolism in derivatives from the neural crest, the neural tube and the epibranchial placodes.

Acetylcholine↗

PubMed1984

Acetylcholine metabolism in rat spinal cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons.

Acetylcholine metabolism has been studied in sister cultures of E13 rat spinal cord cells cultured for 1 to 3 weeks with or without conditioned medium (CM) from rat skeletal muscle cells. Spinal cord cells grown with CM synthesized and accumulated 3 to 4 times more [3H]ACh from [3H]choline than cultures grown without CM. This effect of CM was accompanied by a comparable increase in CAT activity and could not be mimicked by increasing the density of the spinal cord cultures. A 2- to 3-fold increase in AChE activity was also observed in 2- to 3-week-old CM cultures, whereas the activity of lactate dehydrogenase was identical in cultures grown with and without CM. We have compared the effects of CMs from various non-neuronal cell cultures on [3H]ACh synthesis and storage by spinal cord cultures and by sympathetic neuron cultures. CM by skeletal muscle greater than skin fibroblasts greater than rat heart muscle greater than C6 glioma cells were the most active on both types of neuron cultures, whereas CM from rat brain, L6 myoblasts, mouse 3T3, and PYT21 fibroblasts was inactive on spinal cord cultures and only weakly active on sympathetic neurons. Serum-free CM from skeletal muscle was inactive on both types of neuron cultures. The CM factor active on spinal cord cultures has been purified several thousand-fold by using a four-step fractionation scheme which has previously led to a partial purification of the CM factor involved in the regulation of CAT, AChE, and catecholamine-synthesizing enzymes in sympathetic neuron cultures ( Swerts , J. P., A. Le Van Thai, A. Vigny , and M. J. Weber (1983) Dev. Biol. 100: 1-11). Moreover, a comparison of dose-response curves established with this purified material showed that it exerted its effects on spinal cord and on sympathetic neuron cultures in the same range of concentration. Thus, these results suggest that the same macromolecule is involved in the regulation of neurotransmitter phenotype in both types of cultures despite their different embryological origins.

Acetylcholine↗

PubMed1984

Transport of potassium, amino acids, and glucose in cells transformed by Rous sarcoma virus.

Transport rates of a number of nutrients and ions have been surveyed in chicken embryo fibroblasts that were density inhibited, growing exponentially, or transformed by Rous sarcoma virus. All the transport systems examined displayed changes associated with changes in growth rate. The rate of ouabain-sensitive potassium transport declined in density-inhibited cells, and increased rapidly in response to serum stimulation. This transport system was regulated both by changes in the activity of the transporters and by the number of transporters in the cell membrane. The rate of transport of the amino acid analog alpha-aminoisobutyric acid declined when cells became density inhibited, but also showed alterations in regulation that were associated with malignant transformation. The rate of glucose transport displayed both growth state-related and transformation-specific changes. The increased rate of glucose transport seen in transformed cells is due to an increase in the number of glucose transporters in the cell membrane. Increased glucose transport is necessary for subsequent changes in glycolysis, and temporally precedes some of the changes in activity of glycolytic enzymes.

Amino Acids↗

PubMed1983

A tumor promoter stimulates phosphorylation on tyrosine.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate is mitogenic for normal chicken embryo fibroblasts and also causes these cells to express transiently many properties of cells transformed by Rous sarcoma virus. Since some mitogenic hormones stimulate a tyrosine-specific protein kinase activity, and since the transforming protein of RSV is a tyrosine-specific protein kinase, we have examined whether TPA also stimulates protein phosphorylation on tyrosine. We report here that TPA treatment of normal cells resulted in a very rapid phosphorylation on tyrosine of a protein peak of Mr 40 to 43 kilodaltons. Thus, a similar biochemical activity (tyrosine phosphorylation) is associated with the action of polypeptide mitogenic hormones, Rous sarcoma virus and a tumor promoter. In addition, TPA treatment resulted in rapid changes in phosphorylation of proteins on serine and threonine.

Animals↗

PubMed1983

Plasminogen activator in normal and tumor-bearing mice.

The plasminogen activator levels of a series of murine tumors commonly used in cancer drug screening were determined and compared to the levels found in normal mouse tissues. Tumors high in plasminogen activator included the B16 and colon 26. The Lewis lung and M5076 carcinoma showed an intermediate level of activity, while the plasminogen activator activity in the L1210 leukemia and colon 38 was barely elevated above background. The specific activity of the enzyme (per micrograms protein) in extracts of B16 and colon 26 was three or four times higher than in the most active normal organs surveyed (kidney, lung, brain and intestine). The high level of plasminogen activator activity measured in extracts of the B16 tumor was reflected in a substantial elevation of the levels of fibrin degradation products in the serum of mice carrying this tumor. This result suggests that the tumor-associated plasminogen activator activity is less subject to inhibitory controls in vivo than is the plasminogen activator of most normal tissues, since the total mass of the tumor was far less than the total mass of the fibrinolytically active tissues, and yet the bulk of the fibrin degradation products were caused by the tumor. We conclude that the high levels of plasminogen activator activity which are observed in many human tumors are found only in some of the transplantable murine tumors. Since this enzyme is active at an increased level in vivo in mice carrying the B16 tumor, plasminogen activator may be a suitable target for selective, enzyme-activated chemotherapy with this tumor test system.

Animals↗

PubMed1983

Regulation of enzymes responsible for neurotransmitter synthesis and degradation in cultured rat sympathetic neurons. I. Effects of muscle-conditioned medium.

The enzymatic machinery for neurotransmitter synthesis and breakdown have been compared in sister cultures of newborn rat sympathetic neurons grown for 12-28 days either in the presence (CM+ cultures) or in the absence (CM- cultures) of a culture medium conditioned by rat skeletal muscle cells. Neuron numbers, total protein, and lactate dehydrogenase activities were identical in CM+ and CM- cultures. Choline acetyltransferase activity was 27- to 100-fold higher in homogenates of CM+ than CM- cultures, whereas acetylcholinesterase activity was 2.5-fold lower. The activities of tyrosine hydroxylase (TOH), DOPA decarboxylase, and dopamine beta-hydroxylase were all about twofold lower in homogenates from CM+ cultures. All these effects were also observed in homogenates of sympathetic neuron cultures grown with and without a macromolecular factor partially purified from CM (Weber, J. (1981). Biol. Chem. 256, 3447-3453.). Experiments of mixing homogenates from CM+ and CM- cultures suggested that the differences in each of the enzyme activities did not result from differences in the concentrations of hypothetical reversible enzyme activators and/or inhibitors. In addition, the deficit in TOH activity in CM+ cultures resulted from a decrease in the enzymatic Vmax with no significant variation in the apparent Km's for the substrate and the cofactor. An identical decrease in the Vmax was observed if TOH was assayed under phosphorylating or nonphosphorylating conditions, suggesting that this decrease did not result from differences in the state of enzyme phosphorylation. Immunoprecipitation curves of TOH activity by an anti-TOH antiserum were parallel when performed on homogenates from CM+ and CM- cultures, suggesting a difference in the number of enzyme molecules without detectable alteration of their kinetic properties.

Acetylcholinesterase↗

PubMed1983

Plasmin-activated prodrugs for cancer chemotherapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard.

Many tumors contain elevated levels of plasminogen activator and thus produce elevated levels of the protease plasmin in the milieu of the tumor. We have hypothesized, therefore, that it should be possible to prepare peptidyl prodrug derivatives of anticancer drugs that would be locally activated by tumor-associated plasmin. As an initial test of this hypothesis, we synthesized the peptidyl prodrugs of the anticancer drugs (alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin, AT-125) and N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard) by mixed anhydride coupling of the parent drug with the protected tripeptide, Boc-D-Val-Leu-Lys(Boc)-OH, followed by deprotection with trifluoroacetic acid. The prodrugs showed an increased selective in vitro cytotoxicity for Rous sarcoma virus transformed chicken embryo fibroblasts (which produce elevated levels of plasminogen activator) compared to nontransformed fibroblasts (which produce low levels of plasminogen activator). In the presence of the plasmin inhibitor, p-nitrophenyl p'-guanidinobenzoate at 2 micrograms/mL, the selectivity of the phenylenediamine mustard prodrug was reduced, but there was no effect on the cytotoxicity of the free drug. Furthermore, the prodrug analogue D-valylleucyl-D-lysylphenylenediamine mustard (in which L-Lys has been replaced by D-Lys) was inactive. Finally, the prodrug derivative of acivicin did not display selective toxicity for transformed cells when the cells were cultured in plasminogen-free medium. These results suggest that plasmin hydrolysis is necessary for the activation of the prodrugs. The prodrugs were tested in vivo for antitumor activity. The prodrug of acivicin, like acivicin itself, was inactive against the B16 melanoma, a murine tumor that produces high levels of plasminogen activator. This prodrug was active against the M5076 carcinoma, a tumor that displays only moderate levels of plasminogen activator; however, despite the fact that the prodrug was 2- to 3-fold less toxic on a molar basis than acivicin, there was no evidence of an increased therapeutic index. The prodrug of phenylenediamine mustard was also slightly less toxic than the parent drug, but again there was no evidence for an improved therapeutic index against the B16 tumor.

Animals↗

PubMed1983

Plasmin-activated prodrugs for cancer chemotherapy. 2. Synthesis and biological activity of peptidyl derivatives of doxorubicin.

We have synthesized peptidyl prodrugs of doxorubicin (Dox) designed to be selective substrates of plasmin. Such prodrugs might be locally activated by the elevated levels of plasmin produced near many solid tumors under the action of tumor-associated plasminogen activators. One such prodrug, 3'-(D-Val-Leu-Lys)-Dox, was obtained via a mixed-anhydride coupling with isobutyl chloroformate between the protected peptide Fmoc-D-Val-Leu-N epsilon-Fmoc-Lys-OH and doxorubicin, followed by removal of the Fmoc groups with anhydrous ammonia. Compared to doxorubicin, the prodrug showed about a 7-fold improved selective cytotoxicity against chicken embryo fibroblasts transformed with the Rous sarcoma virus (which produce high levels of plasminogen activator) compared to normal cells (which produce low levels of plasminogen activator). However, the prodrug was a very poor plasmin substrate, and although in vivo tests against the murine B16 melanoma showed that the prodrug was active, the maximum T/C obtained was less than that achieved by doxorubicin even at 25 times the molar concentration of prodrug. Qualitatively similar results were obtained for a far more hydrophobic prodrug, 3'-(Boc-Val-Leu-Lys)-Dox. These results demonstrate that peptidyl prodrugs of doxorubicin designed as plasmin substrates are more selective anticancer agents in vitro than doxorubicin itself but that the bulky anthracycline moiety probably prevents efficient plasmin-catalyzed conversion to the active parent drug, so that, in their present form, these drugs are not potent enough to allow a determination as to whether or not they are more selective in vivo.

Animals↗

PubMed1983

Phosphotyrosine-containing proteins and expression of transformation parameters in cells infected with partial transformation mutants of Rous sarcoma virus.

We have examined the phosphorylation state of five proteins known to become phosphorylated on tyrosine during transformation by Rous sarcoma virus by using cells infected with a panel of partially transforming mutant viruses. Situations of viral mutant and growth temperature were found in which phosphorylation of some proteins occurred more extensively than that of others, indicating that mutations in the src gene had affected the specificity of pp60src for some of its substrates as well as affecting the activity of the enzyme. To obtain insight into the biological functions of these phosphorylations, comparisons were made between the degree of phosphorylation of these proteins and the expression of various indicators of the transformed phenotype. The data suggest that phosphorylation of proteins l, p, and q (Mr of 46,000, 39,000 and 28,000, respectively) is not sufficient to induce changes in adhesiveness, hexose transport or morphology. The phosphorylation of protein p or l or total phosphotyrosine content correlated well with the production of plasminogen activator, and the phosphorylation of proteins l and q correlated well with increased hexose transport. However, even when good correlations were observed, significant exceptions were sometimes noted. It thus remains possible that some phosphorylations on tyrosine observed in Rous sarcoma virus-transformed cells are not causally related to the expression of the measured parameters of transformation.

Animals↗